β-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the golgi complex, shows homology to β-adaptin

Cell, Vol. 64, 649-665, February 8, 1991, Copyright © 1991 by Cell Press

13-COR a 110 kd Protein Associated with Non-Clathrin-Coated Vesicles and the Golgi Complex, Shows Homology to 13-Adaptin Rainer Duden, Gareth Griffiths, Rainer Frank, Patrick Argos, and Thomas E. Kreis European Molecular Biology Laboratory D-6900 Heidelberg Federal Republic of Germany

Summary We have cloned and sequenced I~-COP, a peripheral 110 kd Golgi membrane protein. ~COP shows significant homology to l~-adaptin. It is present in a membrane-bound form and in a cytosolic complex of 1314S, with a Stokes radius of ~10 nm and an estimated Mr of "'550,000. By immunofluorescence labeling, ~COP is associated with the structures of the Golgi complex. Immunoelectron microscopy has localized 13-COP to non-clathrin-coated vesicles and cisternae of the Golgi complex. These coated vesicles accumulate in rat liver Golgi fractions treated with GTPyS and strongly label for ~COR Our data suggest that B-COP is a component of a coat associated with vesicles and cisternae of the Golgi complex. Introduction

The Golgi complex, flanked by membranous networks on its cis and trans sides (Lindsay and Ellisman, 1985; Griffiths and Simons, 1986; Rambourg and Clermont, 1990), is a key organelle where processing and sorting of newly synthesized proteins occurs (for reviews see Farquhar, 1985; Kornfeld and Kornfeld, 1985; Pfeffer and Rothman, 1987). Further characterization of these compartments was obtained when biosynthetic membrane traffic was blocked at reduced temperatures. Incubation of cells at 15°C leads to accumulation of newly synthesized membrane and secretory components in a reticular compartment intermediate to the endoplasmic reticulum (ER) and the cis side of the Golgi complex (Saraste and Kuismanen, 1984). This 15°C intermediate compartment may be involved in recycling of KDEL receptors and retrieval of ER resident proteins (for review see Pelham, 1989). Biosynthetic membrane transport can also be blocked at the trans side of the Golgi complex, in the trans Golgi network (TGN), by incubation of cells at 20°C (Matlin and Simons, 1983). Membrane traffic from the ER to the Golgi complex and from the Golgi complex to the different final cellular destinations is believed to be mediated by carrier vesicles (Palade, 1975). Two populations of coated vesicles mediate biosynthetic membrane traffic between the different membrane-bounded compartments. Clathrin-coated vesicles carry proteins to endocytic organelles (for reviews see Griffiths and Simons, 1986; Brodsky, 1988) and secretory granules (Orci et al., 1985; Tooze and Tooze, 1986). Their major coat components comprise the clathrin heavy (180 kd) and light chains (20-28 kd), which are linked to

the TGN or plasma membrane through adaptor complexes HA-1/AP1 or HA-2/AP2, respectively (Pearse and Robinson, 1984; Keen et al., 1987; Robinson, 1987; Ahle et al., 1988). These adaptor complexes contain a number of components, including the 100-120 kd a-, 13-,and y-adaptins as well as proteins of '~50 kd and ~,20 kd (for reviews see Morris et al., 1989; Keen, 1990). A similar set of proteins, namely, or-COP (160 kd), lY.COP (110 kd), y-COP (98 kd), and 8-COP (61 kd), is associated with the non-clathrin-coated vesicles, which have been suggested to be involved in intra-Golgi transport and transport from the ER to the Golgi complex (Orci et al., 1986; Malhotra et al., 1989; Serafini et al., 1991). The prevailing evidence suggests that Golgi-derived non-clathrin-coated vesicles act as bulk carriers (Orci et al., 1986; Wieland et al., 1987; Karrenbauer et al., 1990), whereas clathrin-coated vesicles carry a selective cargo of membrane proteins (Pearse and Bretscher, 1981; Brodsky, 1988). Brefeldin A, a fungal metabolite, causes rapid redistribution of Golgi proteins into the ER and disappearance of the Golgi complex (Misumi et al., 1986; Fujiwara et al., 1988; Doms et al., 1989; Lippincott-Schwartz et al., 1989). These effects are fully reversible (see, for example, Lippincott-Schwartz et al., 1989). It has been suggested that disappearance of a distinct Golgi complex upon treatment of cells with brefeldin A is a consequence of halting the anterograde membrane transport in the recycling pathway between the Golgi complex and the ER, without disturbing retrograde membrane traffic (Lippincott-Schwartz et al., 1990). While the point remains to be proven, it appears clear that the pharmacological perturbation of this organelle, together with its rapid breakdown at the onset of mitosis (Lucocq et al., 1989) and rearrangement upon treatment of cells with microtubule-disrupting drugs (Ho et sl., 1989; for review see Kreis, 1990) or reduced temperature (Griffiths et al., 1989), demonstrates that the Golgi complex has a very dynamic structure (see also Cooper et al., 1990). We have previously identified and partially characterized a peripheral membrane protein of Mr 110,000 with a monoclonal antibody (MAb), M3A5. This 110 kd protein is present on the cytoplasmic face of Golgi membranes, and it cosediments with taxol-polymerized tubulin in vitro (Allan and Kreis, 1986). The distribution of the 110 kd protein changes rapidly and dramatically upon treatment of cells with brefeldin A. The 110 kd protein apparently dissociates within 30-60 s from the Golgi complex, preceding all other morphological changes of this organelle induced by the drug (Donaldson et al., 1990). Thus, the interaction of the 110 kd protein with Golgi membranes appears to be dynamic and regulated in vivo. Brefeldin A may affect the Golgi complex in various ways; it may either directly interfere with critical events in membrane traffic between the ER and the Golgi complex (e.g., budding or fusion of carrier vesicles) or perturb an essen(ial skeletal scaffold associated with the Golgi complex and involved in preserving the structure and identity of this organelle.

Cell 650

D g

1

2

3

11

21

-,ul

31

Figure 1. Affinity Purificationof 13-COPwith MAb M3A5 A 40% ammoniumsulfateprecipitatedfractionof rat livercytosol(lane 1)was usedfor immunopurificationof p-COPwith MAb M3A5(lane2). ~COP was further purified by preparativeSDS-PAGEand electroelution fromthe gel band(lane3). Lanes1-3 showCoomassisbluestaining and lanes 1'-3' the corresponding immunoblotwith M3AS.M3A5positivebandsof highermobilitythan I~-COPcorrespondto proteolytic fragments. ArrowheadsindicateI~-COP.Molecularweight markerproteins are indicatedon the left (from top to bottom: 205, 116,92, and 66 kd).

In this study, we characterize the Golgi complex-associated 110 kd protein at the molecular level. This protein has been localized to non-clathrin-coated vesicles and cisternae of the Golgi complex by immunoelectron microscopy. The cDNA sequence predicts a novel protein of Mr "u107,000 with significant homology to 13-adaptin, a component of the clathrin adaptor complex. This 110 kd protein is a major component of the coat of non-clathrincoated vesicles of the Golgi complex (see also Serafini et al., 1991). Therefore, and since it may also play an important role in regulating the structural identity of the Golgi complex, we suggest this protein be called 13-COP (for coat protein).

Results cDNA Cloning of I~COP ~COP was cloned by screening of a rat liver cDNA expression library with oligonucleotides derived from partial protein sequences. I~-COP was immunopurifled from a high speed supernatant of a rat liver homogenate using MAb M3A5 (Figure 1; see also Experimental Procedures). This fraction greatly enriched in I~-COP was separated by SDS-PAGE, the 13-COP band excised, and protein electroeluted. Approximately 500 pmol (,~60 I~g) of pooled pure I~-COP was separated again by SDS-PAGE, and the protein in the gel band was digested with trypsin. Tryptic peptides were purified by HPLC and sequenced (for details see Experimental Procedures). Eight peptide sequences were obtained (see underlined regions in Figure 2b). Two degenerate oligonucleotides derived from the peptide sequences were used to screen a rat liver cDNA expression library (for details see Experimental Procedures). All eight clones positive with both oligonucleotides cross-hybridized under stringent conditions. One of the

clones, number 70, produced a fusion protein of 205 kd recognized by M3A5. All clones appeared to be derived from the same mRNA as seen by restriction mapping and partial DNA sequencing. A full-length open reading frame was reconstructed from restriction fragments of two clones, 20 and 81, in the plasmid pGEM, using the internal BamHI restriction site (Figure 2a). This construct was transcribed and translated in vitro (for details see Experimental Procedures). The in vitro translation product reacted with M3A5 and comigrated with 13-COP from rat liver on SDS-PAGE (data not shown). The entire coding sequence of 13-COP predicted from this construct revealed an open reading frame of 2862 bp (Figure 2b). The sequence surrounding the first ATG codon in the open reading frame, GGAACCATGA, conformed well with the consensus sequence, GCC(A/G) CCATGG, for eukaryotic translation initiation (Kozak, 1987). Two nonsense codons were found upstream of the first ATG. Thus, this first ATG is probably used in vivo. The open reading frame contained the coding sequence of all eight tryptic peptides (see underlined regions in Figure 2b). Each of the peptides was preceded by a lysine or an arginine, as expected for tryptic fragments. No polyadenylation signal was detected in the 3' noncoding region, presumably because the available cDNA did not cover the entire length of the I~-COP mRNA. In Northern analysis on poly(A)+ RNA from rat liver and HeLa cells, a single band of ,~3.4 kb was identified (not shown). The cDNA of 13-COP predicts a novel protein with an estimated molecular mass of 107 kd (Figure 2b). There are no hydrophobic stretches long enough to span a lipid bilayer. This is consistent with our previous biochemical characterization of 13-COP(Allan and Kreis, 1986). Neither internal repeats nor recognition sequence motifs for acylation could be detected in the sequence. Several potential phosphorylation sites were found in the I~-COP sequence by search in the PROSITE data library, including sites for protein kinase C and cAMP-dependent protein kinase. Amino acids 879-882 conform to a possible consensus sequence, (T/S)PX(K/R), for phosphoacceptor sites of p34 cdc2 kinase (Moreno and Nurse, 1990). In fact, in Xenopus laevis the I~-COP homolog is cdc2-dependently hyperphosphorylated in meiosis, which appears to lead to its dissociation from membranes (Allan et al., 1988; Allan, Felix, Karsenti, and Kreis, unpublished data). It is not known at present which of these phosphoryl~.tion sites are used in vivo. Protein sequence homology between 13-COPand I~-adaptin in the N-terminal domains (~500 residues) was indicated by searching the GenBank and EMBL data libraries using the quick but not always sensitive TFASTA algorithm (Pearson and Lipman, 1988). Sequence identity was only 17% over about 450 aligned residues. Common features also included hydrophobicity plots (not shown). A more sensitive technique used to confirm distant relationships (Argos, 1987) was then applied to pairwise comparisons of I~-COP with all known adaptin sequences, including human 13-adaptin (Ponnambalam et al., 1990), mouse a-adaptin A (Robinson, 1989), mouse y-adaptin (Robinson, 1990), and a yeast sequence strongly related to

110 kd Adaptin Homolog on Non-Clathrin-Coated Vesicles 651

0

1

2

3kb

i

i

i

i

a

BC

v 20 70

Ba B c A o

I

I

I~g

B

I I

Ao X

I

I

A

I

Hg

I

S

H

i

I

I

BaE

I1,~

I

I

81

!

I

I

b 1

CTT CCC GACC CCCAC C CC CACGGAC-C CAGATTTAC CT GGCACCTTTCGTCCTTTTCATCTTGTTTTGAGT~GGTTATAAACAGGAACCATGACCGCAGCTGAGAACGT GTGCTATACGTT 1 M T A A E N V C Y T L

121 12

A A T T A A C G T G C C A A T GGACTCAGAAC CCC CTT CTGAAATCAGCTTAAAAAAT G A C C T G G A C ~ C 4 2 G A T G T C ~ T C A A A G A C T G A A C ~ ~ T I N v P .M..D.A.E...P...P..S...E...Z..S...~...K.F..D " L ~ K G D v K S K T E A

K

K

V

z

241 52

TGG GGAAAAGCTT CCTGGACTCCT GAT GACGATCATTCGTTT CGTGC TGCCTCTT C A G G A T C A C A C C A T C A A ~ A Z T T G C T T C T G G T ~ T ~ T ~ C ~ G E K L P G L L M T I I R F V L P L Q D H T I K K L L L V F W E

I

V

P

361 92

TGGGAGGCTCTTACAT GAAATGATTCTTGTGTGTGAT GCATACAGAAAGGATCTCCAC, CATCCTAATGAGTTr ATTCGTGGATCTACTCTTC G T T ~ C ~ C ~ T T ~ G ~ G A G R L L H E M I L v c D A Y ~ A..D..~...O......P...N...E..F...I...R.A..S...T ~ R F ~ C K L K E A

481 132

ATT GC T GGAAC CT CTGATGCCTGC TATCCGTGCTTGTTTGGAACATC G T C A C A C ' C T A T G T T A C - G A G A A A C G C T G ~ ' F T T G G C C A T C T A C A C C A T C T A C A G A A A T T T T C ~ A T A C C L L E P L M P A I R A C L E H R H S Y V R R N A V L A I Y T I Y R N F E N L I P

601 172

TGATGCTCCTGAGCTGATACATGATTTTC TGGTAAAC GAGAAGGATGCAAGCTGCAAAAGAAATGCATTTATGATGCTCATTCATGCAGATCAGGATCGAGCT TTGGATTATTTAAGTAC D A P E L I H D F L V N E K D A S C K R N A F M M L I H A D Q D R A L D Y L S T

721 212

ATGTAT TGATCAAGTTCAGACATTTGGAGACATTCTACAGTT G G T T A T T G T T G A A C T A A T T T A T A A G G T C T G T C A T G C T ~ T ~ T ~ C ~ C A T ~ G ~ G T A ~ T A T ~ C I D Q V Q T F G D I L Q L V I V E L I Y K V C H A N P S E R A R F I

I

Y

N

841 252

CTTACTGC~JGTCATCTAGTCCTGCTGTAAAATACGAAGCT GCTGGGACAC T G G T G A C A C T G T C A A G T G C T C C A A C T G C A A T A A A G G C T G C T G C T C ~ J G T G T T A C A T ~ T ~ A ~ L L Q S S S P A V K Y E A A G T L V T L S S A P T A I K A A A Q C Y I D L I

I

K

961 292

GGAAAGTGATAACAATGTAAAGC TCATTGTCCTGGAC CGATTGGTAGAACTAAAAGAGCATCCTGCTCACGAGCGAGTCCT CCAAGATCT GGTCATGGACATCCTACGAGTACTGAGTAC E S D N N V K L I V L D R L V E L K E H P A H E R V L Q D L V M D I L R V L S T

1081 332

ACCAGACCTAGAAGTGCGCAAGAAAAC GCTGCAGTTAGCACT GGATC TTGTCT CATCTAGGAATGT TGAAGAGTTGGTTATT GT CT TGAAGAAGGAAGTAATTAAAACAAATAAC GTGTC P D L E V R K K T L Q L A L D L V S S R N V E E L V I V L K K E V I K T N N V S

1201 372

TGAGCATGAAGACACT GACAAATACC GACAGCTTC TC GTGCGAACACT GCATTCCTGC TCTGT CC GATTTCCAGATATGGCTGCAAATGTTATTCCTGTGCTAAT GGAAT TTCTCAGTGA E H E D T D K Y R Q L L V R T L H S C S V R F P D M A A N V I P V L M E F L S D

1321 412

CAGTAATGAAGCAGCAGCT GCTGATGTCTTGG~JGTTTGT GCGTGAAGCCATTCAGC G C T T T G A ~ T S N E A A A A D V L E F V R E A I Q R F D N

1441 452

TGT C A A G A T T T A T C G A G G A G C A C T G T G G A T C C T G G C - C G A G T A C T G C A G T A C T A ~ G A A G A C A T T C A G A G T G ~ ~ G ~ V K I Y R G A L W I L G E Y C S T K E D I Q S V M T

1561

AGA~TAA~GAAGCTGGCGAGTTAAAAC

1681 532

ACCTAC CAAGAAAGAAGAGGACAGACCACCCTTGAGAGGATT CCTCCTGGATGGAGATTTCTTTGTTGC TGCCTCCCTT C ' C C A C A A C T C T G A C C A A G A T T G C A T T A C G ~ A ~ T A G ~ T T P T K K E E D R P P L R G F L L D G D F F V A A S L A T T L T K I A L R Y V A L

1801 572

GGTTCAC~GAA~GCAAAAGTCTTTTGTTC-CTGAGGCTATGTTGC V O E K K K Q N S F V A E A M L

1921 612

CCGAATTICcCTGTGcCTcAAGGTCTTATCTGAATGCTCACCTTTAATGAATGACATTTTTAATAAGGAGTC~AGACAGTCTCTTTccCAAATGTTGTCTGCCAAACTCGAAGAAGAGAA R I S L C L K V L S E C S P L M N D I F N K E C R Q S L S Q M L S A K L E E E K

2041 652

AcTATCCCAAAAGAAAGAAT~TGAAAAC~AGGAATGTAACAGTACAGCcTGATGAccC~ATTT~CTTCATc.CAACTAACT~T~~cT~G~A~c~cT

2161 692

GAGTTTGT T G G C A G C G A T G G G T A A C A C T C A G A G G A A A G A C - ~ C A G A C C CCCTGGCGTC CAAACTCAACAAGGTCACTCAGTTGACAGGTT T C T C T G A T C C A ~ A ~ G ~ T A S L L A A M G N T Q R K • A A D P L A S K L N K V T Q L T G F S D P V Y A

2281 732

TGT T C A T G T C A A T C A G T A T G A T A T T G T C C T G G A T G T T C T T G T % X ~ T A A A C C ~ C C A G T G A T A C T T T C,C A G A A C T G C A C A T T A G A G T T A G C T A C T C T A G G G ~ T ~ T C G ~ V H V N Q Y D I V L D V L V V N Q T S D T L Q N C T L • L A T L G D L K L V ~ K .......o.o..

2401 77~

ACCATC T C C T T T G A C T C T T G C T C ~ T c A T G A C T T T G c G A A T A T T A A ~ c C A A T G T C A A A G T A G C A T C A A C A C ~ T G G A A T A A T T T T C G G C ~ T A T A G T T .~..A..~..~..A.L.~...~......~..f...~.~..A..L* ~ v ~ v * s ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ v

2~1 8~

~

2641 852

T A ~ T GACAGTTAACACCAACGTGACT¢~.CC TGAATC~TTATTTACAGCACATC C T C ~ G T C C A C C A A C A ~ T K V T V N T N V T D L N D Y L O H I L K S T N M K

2761 892

GGCAGCCAAc~TATATGCTCGCTCTATATTTGGA~TGCAcTTGCAAAT~TCAGCATTGAGAAGCCAGTTCACCAGGGACCAGATGcTGCTGTTACTGGCCATATAAGAATTCGTGC

2881 ~32

AAAAAGTCAGGGAATGGCCTTGAGCCTTC~C~A~TCAACCTGTCTC~A~TAGTCTCTA~T~C~C~T ~ s ~ ~ ~ ~ ~ s .~..~.A..~..~...~..A.s...q..~..~..A..s..~. *

3001

TGATTGGACTCCGACGTTTTTGTACTCTTTCATGCTTTATGTTTGTAGAATTTGAGTTCATGCTGAATTCATT

L

s

A

s

` ~

A

Q

~ ~

N

= ~

L

~ c

Y

v

A

` v

R

~ ~

S

~ s

I

T R

T M

~ C L I

E

L

= ~

F

1

G

~ ~

E

c ~ * ~ ~ ~ ~

D

A

L

A

~ ~

N

~ V

C ~ C C A ~ E K M L E

V

CAGAAGAAGAAATAACTGTTGGGC C C G T T C A G A A A T T G G ~ A ~ T

R

R

S

C L

P

~

* ~ ~ ~ o

V

S

I

s

c ~

E

~

~ *

K

o

~

a •

P

~

~ c

V

~

H

C V

~ T T A T ~ C ~ T M I Z.

K

R

T

~

= ~

~ * ~

C

Q

~

~ L

G

~

~

~ r

G ~ T P

P

~

D

~

~

= ~

C ~ ~ K

A

A

~

I

c

T

~

~

w

~

~

T A

~ L

A S

~ G

~

T ~

C

C~GA P D

S

~

D

~

T

D

~

~AG

D

GTA~ V D

~

~

E

A

Y

TAT G A T G T C T C T C ~ G ~ G C ~ ~ v ~ ~ *

= ~

V

T

C~T ~TA~TC P I V E S

G ~ C ~ A ~ A ~ C ~ T ~ A C T

P

T

T A ~ T C F H A I K

~ T ~ G E I

TTATGGCAACCATCCTTCATTTGGGA~CTC~T~CT~GC~T~C~T M A T I L H L G K S S L P K K

.K..E...s...E..K..A..~..v..A.v..q.A..D.p "

K

~ L

n

~

G

H

~ ~

~

Y

G ~ T ~ T C G F M

I

R

~

I

~

R

~

A

T~T~A~T~GC

Figure 2. cDNA Sequencing of I~-COP (a) Schematic representation of cDNA clones 20, 70, and 81. The structure of the ~-COP cDNA is represented on top (A = Accl, Ap = Apal, B = BamHI, Ba = Ball, Bc = Bcll, Bg = Bglll, E = EcoRI, H = Hpal, Hg = Hgal, S = Sspl, and X = Xbal). The open and filled arrowheads mark the start and end, respectively, of the I~-COP open reading frame. (b) Nucl~tide and deduced amino acid sequence of I~-GOR Underlined regions indicate matches of partial protein sequences of 13-COP with the cDNA-deduced [3-COP protein sequence. Dotted lines indicate the six peptides used for raising antibodies. The asterisk denotes the stop codon.

Cell 652

a

900 ]

~ooI b !)" 8OO q

700 4

>

700

I

600 ]

/ /

OO0 -

/

,/

/

/

t 5OO ]

~j¢

400

4O0

300

300

200

IOO

",/~/ .~

/ i

>,

100

200

/;

I

300

400

i/.

/,

I00

/~/

//

,,,,,

600

700

800

900

ttBADA

• "~ "~ "

100

"

200

400

.TOO

600

/,

.

/I

/

/,

300

/

J

200

" 500

, /'1

/ /

I

j

t

,

5OO

/

,/

700

800

900

MAADA

Figure3. Homologyof 13-COPwith 13-Adaptin (a) Homologysearchmatrix(Argos, 1987;Rachidet al., 1989)between~-COP(RCOP110)and human13-adaptin(HBADA).The searchwindowlengths rangedfrom 5 to 35 in stepsof 2. The searchpeaksare plottedoverthe entirewindowlength,with the largestvaluedominatingwhenoverlapoccurs from the multilengthprobes.The peakvalues(S) are scoredas a numberof standarddeviations(o) abovethe matrixmeanfor eachwindowlength. For this comparison,blackenedbars indicate5.4 a ~ S ~ 6.2 a; thick but lighter lines referto 4.7 a ~ S ~ 5.3 a; and thin lines signify 3.9 a ~ S 4.6 a. The path correspondingto the alignmentshown in Figure 4 is delineatedby arrows. (b) As in (a) exceptfor humanI~-adaptin(HBADA)and mousea-adaptinA (MAADA),with blackenedbars referringto 4.6 a ~ S ~ 5.2 a, thick but lighter lines as 4.2 a ~ S ~<4.5 a, and thin lines indicating3.8 o ~ S ~ 4.1 o.

~-adaptin (YAP80; Kirchhausen, 1990). All the adaptins were also compared with each other. It was clear from the search dot plots (Rechid et al., 1989) that the N-terminal halves (approximately the first 500 residues) of the aforementioned sequences were alignable. The search plots of I3-COP and human 13-adaptin, and of mouse a-adaptin and human I~-adaptin, are shown as examples (Figures 3a and 3b). Significant standard deviation (a) relationships (Rechid et al., 1989) in their N-terminal regions were obtained from the following pairs: human and yeast I~-adaptin at 8.4 a, mouse ct-adaptin and 7-adaptin at 8.7 a, human l~-adaptin and mouse a-adaptin at 3.9 a, and finally 13-COP and human ~-adaptin at 3.2 a (Figure 6), all above the 99% confidence level. In the pairwise comparisons, significant homology of I~-COP was detected only to I~-adaptin. The multiple alignment shown in Figure 4 was determined by first aligning the two closely related pairs mouse ~- and y-adaptin and human and yeast 13-adaptin, and then using the human I~-adaptin sequence to align the two close families and add I~-COR The method of Vingron and Argos (1989) (with settings of 15 and 1 for the respective initiating and extending gap penalties) yielded the final alignment generally in accord with the pairwise search plots. The relationships between 13-COP and the human and yeast I~-adaptins (Ponnambalam et al., 1990; Kirchhausen, 1990), as well as the mouse ~- and y-adaptins (Robinson, 1989, 1990), in their N-terminal domains are illustrated in the tree of Figure 6, where 13-adaptin is seen to be about equidistant from I~-COP and y- and a-adaptins. The delineable sequence relationships of all the adap-

tins and I~-COP suggests a common basic main-chain fold in their N-terminal domains. In the multiple alignment of Figure 4, two lysines and an aspartate (K44, K75, D108 in [}-COP) are fully conserved. A strong consensus region is found at the C-terminus of the alignment, namely, WI(L/I)GEY. The fully conserved residues and the consensus motif may individually or in concert act as general determinants in the overall binding properties of 13-COPand the adaptins. No significant relationship between the C-terminal regions of I~-COP and any of the corresponding adaptin segments could be found. However, relationships among the C-terminal halves of the adaptins were detected. Although these homologies are much weaker, they do show relative relationships similar to the N-terminal domains. For the C-terminal segments the following ..standard deviations were obtained: human and yeast I~-ad~ptin at 4.5 a, mouse a- and y-adaptin at 3.6 a, human a- and mouse 13-adaptin at 2.6 a, and human 13-and mouse 7-adaptin at 0.8 a. Though the latter pair is not matched significantly, it shows a consistent evolutionary pattern with the N-terminal segments (see Figure 6). Figure 3b illustrates the human I~- and mouse a-adaptin C-terminal search plot, and Figure 5 shows the alignment resulting from the trace indicated by arrows in the search matrix. The adaptin C-terminal spans vary greatly in length, with a range of 232 to 514 amino acids, whereas the N-terminal regions vary only between 459 and 511. The protein sequence of I~-COP was also compared with known sequences of sec mutants of Saccharomyces cerevisiae (sec2, sect2, sec15, sec18, sec21, sec23; not

110 kd Adaptin Homolog on Non-Clathrin-CoatedVesicles 653

¥A£80 HBADA RCOPI10 MAADA MGADA

....................... ..................... MY MPAVSKGDGMRGLAVFISDZRNCKSKE MPAPIR ...... LRELIRTIRTARTQA

8BAD& RCOPII0 MAADA HGADA

E VK~V~AAMT E LK~V~IMIL~G~E .... ~LLFIFL RNVA~LLYMEN

YAP80 HBADA ~COPII0 14AADA MGADA

YAPS0 HBADA RCOPll0 HAAOA MGADA

SSSPA KAQEPPES QVATNTET

HGADA

HBADA RCOPII0

HAADA HGADA

YPAEFG0~-: V

O -

A N IRIS I K~KG

Dl~]& L D S ' S K K

C~IRISlSLEIEE'- . . . . . . .

CM -TDNLELIEIKLVI~LIYLM~YA~S R FVLPLQDH~I~KLLLVFWEIVPKTT VN -LLS EE¥~E~0~I'~]FISVLV.SES~LI LK LIA OKF~D~R~GIYLIGAMLLLDERQ~VHL

A R LB~ EM R I N MT

L V P V Kr'E-~L $ D 0- - Y ALFC D E PC I I ~ L R Kv CsL K HDEE$D P P E 01-v-I~'R TI'A-]-I'¥ MGCIRVDEIT L L L°LI4 - - P A I R A C L E S R E S ¥ M G A F AA D I P R I LVA G D S MD S MCRDLAGEVEKLLKTSNS¥--

V

EV

--

L S,,III, L . E F L E L L , E O S

--

YEA>LVTLSS&PTA[IIEAA& QHSNADK']NAILFETISLIIIIBYD --EVGNAILYETVLTIMDIK

N

ERVLQDLVMDI

K 0 EIIIK V F F - - V[-](I¥IID P I ¥ R. . . . . . . . . V L S T PI-DIL E

[YIVIA LIT S L -

-

g RE

m

I[~]L L L S RE S S ~L R L °lI]S Y F F-K R-

PEI

Z E Y E DE I ~ I¥

E NS"

TV Q t D HNAV

R S t I V DC

LIKI- D L

VS

VL L

t' , F R I P

.LLDI"~. H

R K[-L']V

LALP¥

D T K~EC

L'

IJKIRRAME

IVVl D FRKYP L E F VLR.JE A.~AI Q R F D

KYESIIATL~CENLDSL E DARAA IL R N L I[~]E K M L E V F ll[~II K S V K I Y R G & L -

VBELIQLITNSVEM

A ¥ T f ~ o RFL1-

YK

152 156 1"/0 159

209 198 226 216

252 287 270

OE, .0

2,, 307 346 325

A S g AINII A ° V L & v S S El.IV EELVI

E°

,S

IRGMME

V~MLLQ ATD Ir-DII . . . . . . . . M S R K S~l~--~J~A ~ G N L V L D E DISIV ~ H D C~]A V ~ " ~ D I " L ~ E~ F GV D V RD V E K g V I E T N N V S E H E D T D K ¥ R Q L L T L B S C S V R F P D MAA N - V I P VILIM E FILLS D S H E X A ~. A ,mLE,,m . . . . . . . . , , i , , , i E YFLDSCE ........ PEFEADCASGI~IFLA E Y A - P['~K R W H I DITII M R VILIT T A G S

L~vw , R v L °lilY T N R D D V

84 98 i13 104

v

IVLDRLVE HRETNLR NNDKNIR

S F AI-I~Iv N G N

4v

257

L~-~A E E E T L P R I L E

L EK D I 14 ][ R R E K TL~Qr~A L D

39 60

247

, , , , o

IIL~ESDNEVKLVRACINIQI-£1GQF RVLAIINJIILIG F

35

~oo

A~MM I D K ¢ C I

SIC RVTPR R F I~CIYNL V C LET AVEGRL ..... S AMNDI

LE, CYID -PN SG

F L~R N Vr~v RNI LKEePAB

R N S K

TTSVV~QHYLDTHEMI

TA NE TEWG IF ............. I DC -SNYN~K~DREA D¥ ST I° V TFGD ....... ILOL V IVE~IYKVCBANPSER RIVS AST L °¥TYYFVPAPWLSVEL~RL QCYPP~E~A GYSPEHDVS--GISDPFLQVRI~RL~RILGRND~DS

I V°

Y VC

0,~¥[~1C

ALL Dl~lD ' =E ' ° H Rr'~- - L GGLS S I L E D I V K A ~ S S S I P ] E I ~ I V AIA1L E T[-~¥ S I H~I~]K N A N M - D~VAE HDINAQH~EDQL F L D S LRDI'L--]I...~°S,I~I.L~V*.I~I**L~s~IzSI~Is.~.S . . . . . . . . . . . . . . . I ~ D : I ¥ T I ¥ R N~F E[E[L ~ o AIPIE.~L I S D F L - - V SlEIK D A S C K R E K FCmL L E[~]¥ K A S P D LI-~F N G T A v v E L - - D S N GIV~V t Ar'A'-'~ S L I T C L C K K NI'P']D ~CIAVH V I R KV P E LME M--F LPA T K N L g N E GML H TS ~ V L[~JT[~]I4 Cr'EIR s L ~ D M L &

YAP80 HBADA RCOPIIO MAADk HGADA

YAP80 RCOPII0

V S~FPDVVN L G~LM~iI HDIDFGEM-

~

I K RW ' ~ - ~ L

EERE~

I L V C -O- A ¥ R KID ' ~LIQ S P EFIRG CELEE A .... ~A~IK BID LIA S R P T F M C I L & Lie C I A N V G S R I.I C Izl K N E H S T Q F V Q GLL~CI~G C M G S S

PQNIEE BADQDRA SLAVSR PQLVRI~KNLIM

YAP80

,

K--

R E A L P FL~]M D~I)]F E S R D E K L 0 I I M A vrNis F v KIDIC E D P P L I R A

YAPS0 HBADA RCOPIIO MAAD& HGADA

HEAD& RCOPII0 MAADk MGADA

K V E A R ¥ K A N~I P T D L 0 E~G V :~- F K S N I T R :~K i iSDQTDs AEN V EC¥ T L i l l e V P H D S E ~' ~'r~E I S L K N D L E K G D V K KS-

364 356 358

,0, 383

417 435 418 ,,,

-

V L

AER CSTK

459 469

L

A [ ~ ] , C B E N[H]v K v G G

L

G N L[~]

511

I

G D ¥ S Q Q P L V Q V A A L ~ C IIG E YIG D L L

487

Figure 4. Sequence Alignmentof the N-TerminalDomains of ~,-COP and Adaptins The sequencesof ~-COP (RCOPl10), 13-adaptinsfrom human (HBADA)and yeast (YAPSO),mouse a-adaptin A (MAADA), and mouse 7-adaptin (MGADA) were aligned. Sequence positionsfor the rightmostresidueof each species are given in the righthandcolumn. Residuesare boxed if three of the five in a given match column are identicallyconserved.

yet accessible through the data libraries), which are blocked at specific stages of secretion (Novick et al., 1980). No significant similarities were detected. M3A5 reacts with both I~-COP and the microtubuleassociated protein MAP2, against which it was raised (see Allan and Kreis, 1986). Therefore, the I~-COP protein sequence was compared with the sequence of MAP2 (Lewis et al., 1988) and other sequenced microtubule-associated proteins, including MAP-U (Aizawa et al., 1990), MAP1B (Noble et al., 1989), and the microtubule-associated motor proteins kinesin (Yang et al., 1989) and dynamin (Obar et al., 1990). No significant sequence similarities were detected. However, lYCOP contains four dispersed copies of a sequence motif, KKEX (X = E,A,V,S), that is also present in 20 closely spaced copies in the microtubule-binding domain of MAP1B (Noble et al., 1989). It is unclear whether this motif coincides with a potential microtubule-binding site of I~-COR

Characterization of Anti-Peptide Antibodies Antibodies were raised against six peptides of ~-COP (dotted lines in Figure 2b), affinity purified, and characterized by immunoblotting and immunofluorescence. One antibody, anti-EAGE, raised against a peptide covering amino acids 496-513, recognized ~-COP in the Golgi complex of various cells and tissues ranging from human to bird (not shown). In addition, a vesicular staining for I)-COP was clearly evident in confocal fluorescence microscopy using anti-EAGE (Figure 7). The immunofluorescence signal of anti-EAGE (but not of M3A5) was completely abolished by preincubation of the antibodies with excess EAGE peptide, thus demonstrating specificity of the labeling (not shown). The other antibodies and M3A5 gave very similar results (data not shown; see Experimental Procedures), confirming the authenticity of the 13-COP cDNA. In.contrast to M3A5, none of the peptide antibodies reacted with MAP2 by immunoblotting.

Cell 654

HBADA MAADA

(460) (512)

RCOPII0

.... DNADELLESFLEGFHDESTQVQLTLLTAIVKLFL AGDPRSSPPVQFSLLHSKFHLCSVATRA/ILLSTYIKFI + * * + + + **

KKP SETQELVQQVLSLATQ NLFP E TKAT I QGVLRAG •* +* ** +

*+

-D SDNPDLRDRGY I YWRLLS TDPVTAKEVVL SE S QLRNADVE LQQRAVE YLT L S SVAS TDVLATVLEE +* + +* +* * * * + ** *

KPLISEETDLIEPTLLDELICHIGSLASVYHKPPNAFVEGSHGIHRKHLPIH MPPFPERESSILAKLKRK ............. KGPGA ........... • * * * *+* *

ASALD +

HGSTDAGDSPVGTTTATNLEQPQVIPSQGDLLGDLLNLDLGPPVNVPQVSSM DSRRDTSSNDINGG .... VEPTPSTVSTPSPSADLLGLRAAPPPAAPPAPVG • + +* * + *** * + ** +*

aYAP 80

+

QMGAVDLLGGG ...... LDSLVGQSFIPS-SVPATFAP .... SPTPAWSSG GNLLVDVFSDGPTAQPSLGPTPEEAFLSELEPPAPESPMALLADPAPAADPG +**+ * * *+ ** * ++

~" .AADA HBADA

*

~ LNDLFELSTGIGMAPGGYVAPKAVWLPAVKAKGLEISGTFTHRQGHIYMEMN PEDIGPPIPEADELLNKFVCKNSGVLFENQLLQIGVKSEFRQNLGRMY--LF +*+ + + +*+ ~ + + + *

*

+*

+

FTNKALQHMTDFAIQFNKNSFGVIPSTPLAIHTPLMPNQSIDVSLPLNTLGP YGNKT SVQFQNF - - - LP TVArHp GDLQT QLAVQTKRVAAQVD GGAQVQQVLN + ** +* ++ * **+ * + * + * VMKMEP LNNLQVAVK - - - NN I DVFYF S C L I P LNVLFVEDGKMERQVF ECLRDFLTPPLLSVRFRYGGTAQSLTLKLPVTINKFFQPTEMAAQDFFQRWK + + * + *+ * * + * * D I PNENELQFQ QL S LP LQEAQK ++ +

I

LATWK *

**

I KECHLNAD - - TVS SKLQNNNVY T I AKRNVE GQDMLY I FKANHPMDAEVTKAKLLGF G SALLDNVDPNPENFVGAG * + +* ** + + + ++ +

Q S LK II +

LTNGIWILAELRIQPGNPN--YTLSLKCRAPEVSQYIYQVYDSILKNQDWSS QTKALQVGCLLRLEPNAQAQMYRLTLRTSKEPVSR/4LCEL .... LAQQF • + + + **++* * *+*+ ** ++ ++ * +* TLQPCCDRCKSRTLNWKKLYCCVESEHTEAT

M

G

A

D

A

Figure 6. Relationships among the N-Terminal Domains of Rat Liver ~-COP and Adaptins The tree shows the approximate relationships among the N-terminal domains of rat liver I~-COP (RCOP110), ~adaptins from human (HBADA) and yeast (YAP80), mouse u-adaptin (MAADA), and mouse 7-adaptin (MGADA). The values given near the curved arrows refer to the closeness of the sequence relationship for a particular pair as a number of standard deviations (o) above a control mean correlation coefficient for matched residues over five residue physicochemical characteristics (see E~xperimentalProcedures; Rechid et al., 1989). For example; human I~-adaptin (HBADA) is related to ~-COP and mouse a-adal~tin (MAADA) at the 3.2 o and 3.9 o levels, respectively.

(973) (977)

Figure 5. Sequence Alignment of the C-Terminal Domains of Human ~-Adaptin (H~DA) and Mouse u-Adaptin A (MAADA) The asterisks indicate identically conserved residues, and plus signs indic=e conserved residues according to the following amino acid groups: (A~I,L,M,C), (W,EH~), (K,R), (Q,E,D,N), (RG), and (S,T).

13-COP Is Associated with Non-Clathrin-Coated Vesicles and Cisternae of the Golgi Complex The distribution of I~-COP was studied by i m m u n o e l e c t r o n microscopy with anti-EAGE on cryosections of Vero cells (Figure 8), NRK cells (not shown), and rat liver (not shown)• Specific labeling on Vero cells (Figure 8, arrowheads), N R K cells, and rat liver was seen preferentially on vesicular structures in the region of the Golgi c o m p l e x and

rims of Golgi cisternae. In s o m e images label was clearly associated with a distinct cytoplasmic coat on the vesicles. Labeling was much reduced toward the center of the Golgi stacks (Figure 8a, arrows). The coated vesicles apparently were derived from Golgi cisternae since buclding profiles with similar m o r p h o l o g y were frequently observed (Figure 8d). None of the endocytic organelles, labeled with 5 nm g o l d - B S A internalized by Vero cells for various periods of time, contained 13-COP (data not shown)• Isolated Golgi stacks from rat liver treated with or without GTPyS in the presence of cytosol (see Experimental Procedures) were also used for i m m u n o l a b e l i n g with antiEAGE. The distribution of 13-COP on these fractions was essentially the s a m e as that seen on Vero cells. ~ C O P labeling was seen preferentially on coated vesicles (di-

Figure 7. Stereo View of the Distribution of I~-COP in Vero Cells Vero cells were labeled with anti-EAGE and rhodamine-conjugatad goat anti-rabbit antibodies. The stereo images are superimpositions of a series of optical sections taken with a confocal beam scanning laser fluorescence microscope (for details see Experimental Procedures). Distinct vesicular structures scattered throughout the cytoplasm are well resolved. Note that the distribution of I~-COP in the region of the Golgi complex is not homogeneous. (The stereo images should be viewed with stereo glasses.) Bar = 5 p.m.

110 kd Adaptin Homolog on Non-Clathrin-Coated Vesicles 655

Figure 8. Localization of Z-COP by Immunoelectron Microscopy on Vero Cells Immunogold labeling of 13-COP with anti-F.AGE (arrowheads) on ultrethin frozen sections of Vero cells is shown in an overview (a) and at higher magnification in areas of the Golgi complex (b-e). Significant labeling for ~COP was seen on Golgi cisternae (arrows in a). ~COP was preferentially found on coated vesicles in the region of the Golgi complex, usually near the rims of cisternae (b, c) and on budding profiles (d, e). Clathrin-coated vesicles (arrow in a; arrow in d) or endosomes (a) were consistently not labeled. CV = coated vesicle; E = endosome; G = Golgi complex; N = nucleus. Bar = 100 nm.

Cell 656

Figure 9. Localization of I~-COP by Immunoelectron Microscopy on GTPyS-TreatedRat Liver Golgi Fractions Immunogold labeling of I~-COP with anti-EAGE (arrowheads) on ultrathin frozen sections of GTPyS-treated rat liver Golgi fractions is shown in an overview (a) and in enlarged areas of the Golgi complex. I~COP was preferentially found on coated vesicles close to Golgi cisternae (arrowheads in a, c--e) and on budding profiles (arrowheads in b, for example). The arrow in (c) indicates a I~-COP-labeledcoated vesicle between two adjacent Golgi cisternae. Labeling on a fenestrated region of the Golgi complex is shown in (d). C = Golgi cisternae; F = fenestrated Golgi membranes. Bar = 100 nm.

ameter ,~70 nm) and buds on cisternae. Since the nonhydrolyzable GTP a n a l o g GTPTS blocks intra-Golgi transport and causes accumulation of non-clathrin-coated Golgi vesicles in vitro (Melancon et al., 1987; Orci et al., 1989), we analyzed w h e t h e r GTPyS also had an effect on

the distribution of I~-COP in isolated rat liver Golgi complex. G o l g i fractions were incubated for 15 min at 3-/°C in the presence of an ATP-regenerating system and 20 I~M GTPTS. Controls without GTPyS were incubated for 15 min at 37°C or on ice. GTPTS increased the labeling for

110 kd Adaptin Hornolog on Non-Clathrin-Coated Vesicles 657

b

C

Figure 10. Localizationof I~-COPand Clathrin on GTPyS-TreatedRat Liver Golgi Fractions Immunogoldlabelingof [3-COPwith anti-EAGE(9 nm gold) and clathrin with an antiserumagainstclathrin light chains (5 nm gold)on ultrathinfrozen sections of GTPyS-treatedrat liver Golgi fractions is shown in an overviewin (a) and in enlargedareas of the Golgi complex (b and c). The coat of the vesicles containedeither t~-COP(arrows)or clathrin (=C"arrowheads).Bar = 100 nm. 13-COP on coated vesicles significantly (see Figure 9). In the controls, fewer labeled vesicles could be detected (not shown). The coat of the vesicles labeled with anti-EAGE consistently appeared different from that of clathrin-coated vesicles (see Figures 8a and 8d). In fact, rat liver Golgi fractions treated with GTPyS and double labeled with anti-EAGE and an antiserum against clathrin light chains showed that vesicles labeled with anti-EAGE did not contain clathrin, and vice versa (Figure 10). ~COP is also absent from clathrin.coated vesicles by immunoblotting (not

shown; see also Allan and Kreis, 1986), and none of the six ~COP peptide antibodies cross-reacted with proteins present in a purified clathrin-coated vesicle fraction (not shown). Thus, we concluded that p-COP is a component of non-clathrin-coated vesicles in the Golgi complex.

13-COP Is Associated with Membranes of the Intermediate Compartment at 15°C and the TGN at 20°C We used temperature blocks of the biosynthetic transport of tsO45 vesicular stomatitis virus glycoprotein (VSV-G) in

Cell 658

Figure 11. Colocalization of I~-COP with VSV-G Arrested in the Intermediate Compartment at 15°(3 and in the TGN at 20°C tsO45-VSV-infectedVeto cells were kept for 2.5 hr at the nonpermissive temperature of 39.5°C (a, b), or were further incubated with 10 p.g/mlcyclohexamide for 2 hr at t5°C to accumulate VSV-G in the expanded intermediate compartment (c, d) or at 20°C to accumulate VSV-G in the TGN (e, f). Cells were then fixed and extracted for 4 min in methanol at -200C and double labeled for immunofluorescence with anti-EAGE (a, c, e) and MAb P5D4 (b, d, f) and rhodamine- or fluorescein-conjugated anti-rabbit and anti-mouse, respectively. Arrows in (c) and (d) indicate patches accumulating at 15°C containing I~-COPand VSV-G.Arrowheads in (e) and (f) indicate an expanded TGN. Labeling for I~-COPand VSV-G was coincident when cells were kept at 150C; at 20°C, however, a number of vesicles containing VSV-G did not label for 13-CORAsterisks indicate uninfected cells. Note that the distribution of ~COP changes at 15°(3 or 20°C independently of whether cells were infected with virus. Bar = 20 I~m.

infected cells to analyze in further detail the intracellular distribution of I~-COP by immunofluorescence. Cells were double labeled with anti-EAGE and MAb P5D4 (Kreis, 1986) against VSV-G (Figure 11). VSV-G is blocked in the

ER at nonpermissive temperature (39.5°C) and transported to the cell surface when cells are shifted to permissive temperature (31°C). When infected cells are shifted to 15°C or 20°C, VSV-G accumulates in an intermediate

110 kd Adaptin Homologon Non-Clathrin-CoatedVesicles 659

43sl

1115sl

1~sI

0.6

I

04

0.2

a,

v

I

I

5

I

I

I

'

I

=

-r

,

10

,

T

|

'

5

FRACTION NO.

b. C.

--"-

Figure12. ~-COPIs Presentin a SolubleComplex The profileof cytosolicproteinfromrat liver(opencircles)or Verocells (filledcircles)sedimentedthrough5%-30%(wt/vol)sucrosegradients and fractionatedis shownin (a).The positionsof markerproteinsare indicated(BSA,4.3S;catalase.11.15S;and thyroglobulin,16.5S).The correspondingimmunoblotsof the gradientfractionsfrom rat liverand Vero cellswith MAb M3A5are shown in (b) and (c), respectively.

compartment (Saraste and Kuismanen, 1984; Schweizer et al., 1990) or the TGN (Matlin and Simons, 1983), respectively. ~COP essentially colocalized with VSV-G in cells kept at 15°C and accumulated in the numerous patches scattered throughout the cytoplasm (Figures 11c and 11d). At 20°C, ~-COP gave a compact staining in the region of the Golgi complex, coincident with the pattern of the "I'GN containing VSV-G (Figures 1le and 11f). Many of the vesicular structures in the cytoplasm containing VSV-G were, however, not labeled with anti-EAGE (Figures 11e and 11f). I~-COP accumulated in the structures enlarged at 15°C and 20°C independent of whether the cells were infected with tsO45-VSV (Figures 11a-11f; asterisks indicate noninfected cells). No significant overlap in the distribution of ~-COP with VSV-G in the ER at nonpermissive temperature was detected (Figures 11a and 11b). The colocalization of 13-COP at 15°C and 20°C with VSV-G suggests that I~-COP associates with the membrane networks on both the cis and trans sides of the Golgi complex in vivo. Treatment of cells with brefeldin A at 37°C led to rapid (<2 min) apparent dissociation of 13-COPfrom these structures expanded at 15°C and 20°C (not shown; see also Donaldson et al., 1990) and from the normal Golgi complex, thus demonstrating a dynamic association of I~-COP with these membranous organelles. No effects on the distribution of VSV-G were detected at these short periods of treatment of cells with brefeldin A. 1[3-COP Is Present in a Soluble Complex Flotation gradient experiments using isolated rat liver Golgi membranes showed that a significant fraction of

13-COP is membrane bound (data not shown; see also Allan and Kreis, 1986). In addition to this membrane-bound form, however, soluble I3-COP was detected both in rat liver and Vero cells. A large proportion (>80%) of I~-COP was solubilized upon homogenization of rat liver and Vero cells in the buffers used to isolate stacked Golgi membranes (Leelavathi et al., 1970; see also Experimental Procedures). This cytosolic form of 13-COPwas characterized by sucrose gradient sedimentation (Figure 12) and gel filtration. By sedimentation in sucrose gradients, soluble 13-COP from Vero cells (Figure 12c) and rat liver (Figure 12b) sedimented in a single peak corresponding to an S value of 13-14S. No monomeric I~-COPwas detected. Less than 20% of I~-COP was detected in the pellet (probably aggregates; not shown). Gel filtration analysis showed that soluble 13-COPeluted in a peak at a position corresponding to an apparent Mr of 700,000-800,000 (not shown) and a Stokes radius of ,,,10 nm. Some soluble I~-COP also eluted in the void volume of the column (<20%). From the data obtained from sucrose gradient centrifugation and gel filtration, a =true" relative molecular weight of ,,,550,000 and a frictional ratio of ,x,1.84 were estimated, using the method of Siegel and Monty (1966) and assuming a partial specific volume of 0.725 cm3/g. Thus, the soluble 13-COP complex is not globular (Tanford, 1961). It is likely that this complex of distinct size contains other proteins in addition to I~-COR The precise protein composition of the complex is not known so far. Discussion

We have cloned and sequenced 13-COR a Mr 110,000 peripheral Golgi membrane protein (Allan and Kreis, 1986), using a rat liver cDNA expression library. The sequence predicts a novel 107 kd protein with significant homology to I~-adaptin. The delineable sequence relationship in the first ,,,500 residues of 13-COP and 13-adaptin suggests a common main-chain fold and function. ~ and 13'-adaptin present in the plasma membrane- and Golgi complexassociated adaptor complex, respectively, are very closely related (Kirchhausen et al., 1989; see also Morris et al., 1989), and the N-terminal domains of these adaptins bind clathrin with high affinity in vitro (Ahle and Ungewickell, 1989; Keen and Beck, 1989). It is thus conceivable that the N-terminal region of I~-COP may interact with a clathrinlike molecule. ~COP has been localized to coated vesicles and cisternae of the Golgi complex by immunoelectron microscopy. These coated vesicles are clearly Golgi derived since numerous budding profiles could be detected at the rims of Golgi cisternae. The coat of these vesicles does not contain clathrin, nor is 13-COP associated with clathrincoated vesicles. These vesicles are identical to the vesicles morphologically characterized by Orci et al. (1986, 1989) and purified by Malhotra et al. (1989), since 13-COP is a major component of the coat of purified non-clathrincoated Golgi carrier vesicles (Serafini et al., 1991). In fact, a nonapeptide sequence of a tryptic fragment of purified ~-COP of rabbit liver Golgi-derived non-clathrin-coated

Cell 660

vesicles is identical to amino acids 481-489 (Figure 2b) of 13-COP (Serafini et al., 1991). Although the coats associated with the clathrin-coated vesicles and Golgi-derived non-clathrin-coated vesicles may prove to be structurally homologous, their selectivity must be different. The adaptor complexes associated with clathrin-coated vesicles selectively interact with the cytoplasmic tails of specific receptors, mediating linkage to the clathrin coat in the budding vesicles (Pearee, 1988; Glickman et al., 1989; Mahaffey et al., 1990). The non-clathrincoated vesicles, on the other hand, are supposed to be the bulk-flow carriers that do not selectivelyconcentrate transport cargo (Orci et al., 1986; Wieland et al., 1987; Karrenbauer et al., 1990). Yet, a set of coat proteins homologous to the clathrin coat may be involved in budding of these non-clathrin-coated vesicles (Malhotra et al., 1989). Indeed, the coat of Golgi-derived vesicles is composed of a set of proteins similar in molecular weight to those of the clathrin-coated vesicles (Serafini et al., 1991). a-COP (160 kd) may be related to clathrin heavy chain, ~COP (110 kd) characterized here is homologous to I~-adaptin,y-COP (98 kd) may be related to another member of the adaptin family, and 6-COP (61 kd) could be similar to the ~,50 kd adaptin subunits. Thus, ~COP may be involved in budding of Golgi-derived coated vesicles. By analogy to the 13-adaptin-clathrin interaction, then, ~COP may interact via its N-terminal domain with a-COP and mediate binding of the coat proteins to the vesicle membrane. It has also been suggested that intersubunit interactions of a- and ~-adaptins, and their interaction with the ,~50 kd and ~20 kd proteins of the HA-2/AP2 complex, involves the N-terminal domains of the adaptins (Keen and Beck, 1989). Therefore, ~COP may also directly interact with other proteins of the coat of the non-clathrin-coated vesicles. No relationship of the C-terminal domain of 13-COPwith any of the adaptins was detected, implying that these regions on the molecules have functions specific to non-clathrin-coated and clathrin-coated vesicles, respectively. However, relationships among the C-terminal adaptin regions were detected, although they were weaker than those among the N-terminal domains. It is likely that sequence divergence in these C-terminal domains corresponds to the variability of molecular interactions in which these domains are involved (Pearse, 1988; Glickman et al., 1989; Mahaffey et al., 1990) but does not preclude a similar fold for the adaptin and I~-COP C-terminal regions. There are many examples of protein tertiary structures with similar topology and related function and yet without detectable primary sequence relationship (see, for example, Rossmann and Argos, 1981; Rossmann, 1987). It may be proposed that rapid dissociation of I~-COP from membranes of the Golgi complex following treatment of cells with brefeldin A leads to an inhibition of budding. A consequence of this would be that forward flow of membrane components will be halted. Since retrograde transport from the Golgi back to the ER continues (LippincottSchwartz et al., 1990), it seems likely that this process, whatever the mechanism, is independent of the function of 13-COR Golgi membranes will thus redistribute into the ER, finally leaving no morphologically distinct Golgi com-

plex. It has indeed been demonstrated by various experiments that the Golgi complex is a very dynamic organeUe (Griffiths et al., 1989; Cooper et al., 1990; Ho et al., 1989, 1990; Lippincott-Schwartz et al., 1990). The immobilization of 13-COP on the membranes of Golgi-derived nonclathrin-coated vesicles by treatment of Golgi complex with GTPyS in vitro, on the other hand, may prevent fusion of these carrier vesicles with the acceptor membranes. Alternatively, I~-COPmay be involved in linking a peripheral scaffold or skeletal framework to membranes of the Golgi complex, including Golgi-derived vesicles. The presence of such a scaffold could regulate docking or fusion events and thus play a role in maintaining structure and identity of the Golgi complex. In this model, movement of membrane within or out of the Golgi complex would rely on local and transient disassembly or removal of this scaffold. The removal of this scaffold would therefore result in the random interaction and fusion of the membranes of the organelles of the secretory pathway, leading ultimately to the disappearance of the individual organelles. Additionally, a potential function of such a membrane scaffold containing 13-COPcould be to maintain the structure and position of the Golgi complex. Its removal, induced by hyperphosphorylation during mitosis or upon treatment of cells with brefeldin A, for example, might result in the dissociation of this structural scaffold from the membranes and induce disassembly of the Golgi complex. It is conceivable that this scaffold interacts with microtubules that maintain the position of the Golgi complex in the region of the microtubule-organizing center (for a review see Kreis, 1990). Whether or not 13-COPis involved in the interaction of such a scaffold with micretubules is unclear so far. The presence of a soluble complex containing ~-COP and the rapid dissociation of this protein from the Golgi complex upon treatment of cells with brefeldin A suggest that there is an equilibrium between a soluble and a "skeletal;' membrane-bound pool. This equilibrium appears to be regulated by GTP, ATP (Donaldson et al., 1991), and probably by phosphorylation of 13-COP (Allan et al., 1988). The effect of GTP on 13-COPmay be mediated by a small GTP-binding protein(s). Phosphorylation of I~-COP appears to be cdc2 dependent in Xenopus oocytes (Allan, Felix, Karsenti, and Kreis, unpublished data), and it is tempting to speculate that it may also directly or indirectly induce the dissociation of this scaffok:l associated with the Golgi complex, leading to disassembly of this organelle at the onset of mitosis. Whether the regulation of association of 13-COP with membranes is mediated through only one or several domains of the molecule can now be studied. It will also be important to identify and characterize the proteins present in the 13-14S complex containing 13-COR since these proteins may play an important role in regulating the function of I~-COR Experlmental Procedures Purification of ~COP

Liversfrom freshlysacrificedSprague-Dawleyratsweremincedwith scalpelsand homogenizedwitha Polytron(Kinematica,Lucerne,Switzerland)in ice-coldbuffer B (20 mM sodiumphosphate,150 mM KCI,

110 kd Adaptin Homolog on Non-Clathrin-Coated Vesicles 661

5 mM MgCI2 [pH 6.7]; 0.5 g/ml buffer) with protease inhibitors (1 mM PMSF, 4 mM benzamidine, and 5 p.g/ml each of TAME, TPCK, and aprotinin [Sigma, Deisenhofen, FRG]). A postnuclear supernatant, prepared by centrifugation at 12,000 x g for 20 rain at 4°C, was recentrifuged at 100,000 x g for 1 hr at 4°C. The supernatant was centrifuged at 200,000 x g for 2 hr at 4°C to obtain a soluble cytosolic fraction (protein concentration 40 mg/ml). This cytosolic fraction was precipitated with 40°/0 ammonium sulfate and the resulting pellet dissolved in one-fourth of the original volume in 50 mM Tris, 100 mM NaCI (pH 7.5) and dialyzed against the same buffer. Aliquots (100 mg of protein per ml) were frozen in liquid nitrogen and stored at -70°C. Aliquots were thawed, adjusted to 2°/0 SDS, boiled for 5 min, and Triton )(-100 was added to 2°/0. For immunopurification Of ~-COP with M3A5 (Allan and Kreis, 1986), 20 p.I of M3A5 ascites fluid and 100 p.I of a 1:1 slurry of protein G-Sepharose beads (Pharmacia LKB) were added to 1 ml of extract and incubated overnight at 4°C. Beads were pelleted at 50 x g and then washed three times with 50 mM Tris, 100 mM NaCI, 0.1% SDS, 0.5% Triton X-100 (pH 7.5) and once with 50 mM Tris (pH 7.5). SDS sample buffer was added, and G-COP-antibody complexes were dissociated by boiling. ~COP and contaminating proteins were separated by SDS-PAGE on 16 cm long preparative 6o/0-12% polyacrylamide gradient gels. Gels were stained with copper chloride, the ~-COP band excised, and protein electroeluted following the procedure of Lee et al. (1987). Approximately 2 p.g of ~-COP was obtained per rat liver (,'~10 g) by this procedure. Protein Sequencing Approximately 500 pmol (,~60 I~g) of purified ~-COP was run on a preparative 60/0-120/0 polyacrylamide gradient SDS-PAGE. The Coomassie blue-stainod I~-COP band was excised, washed with water, and digested for 12 hr with trypsin (10o/0 [w/w]; sequencing grade, Boehringer, Mannheim, FRG). Peptides were separated by reversephase HPLC on a microbora column and eluted with a gradient of 0%-80% acetonitrile in 0.1% aqueous trifluoroacetic acid. Fractions containing peptides were lyophilized and sequenced using a gasphase sequenator with on-line HPLC detection (see Gausepohl et al., 1986). Isolation of cDNA Clones Two degenerate oligodeoxynucleotide hybridization probes with a length of 23 bases were prepared from the peptide sequences EMGTYAT(Q) (amino acids 517-524) and KPEEEIT(V) (amino acids 501-508), oligonucleotides I and 2, respectively. The sequence of oligonucleotide 1 was 5'-GA(AG)ATGGG(ATCG)AC(ATC)TA(TC)GC(ATC) AC(ATC)CA-3', and the sequence of oligonucleotide 2 was 5'-AA(AG) CC(ATC)GA(AG)GA(AG)GA(AG)AT(ATC)AC(ATC)GT-3'. All nucleotides in parentheses were included at that position. Both oligonucleotides are 432-fold degenerates. Approximately 500,000 colonies of a random-primed rat liver cDNA library (kindly provided by Dr. K. K. Stanley) made in the expression plasmid pUEX (Bressan and Stanley, 1987; Luzio et al., 1990) were screened. Oligonucleotides were 32p-labeled with polynucleotide kinase and ['y-32p]ATP. Oligonucleotide 1 was used in the first screening and oligonucleotide 2 in successive rounds. Plating of bacteria and lifts were done by standard techniques (Sambrook et al., 1989). Nitrocellulose filters were purchased from Schleichar & Sch011 (Dassel, FRG). After baking for 2 hr at 80°C, nitrocellulose filters were washed four times for 10 min in 3x SSC, 0.1% SDS at room temperature followed by 1.5 hr in 3x SSC at 68°C to remove bacterial debris. Prehybridization was performed overnight in 6x SSC, 5x Denhardt's solution, 0.05% sodium pyrophosphate, 100 p.g/ml herring sperm DNA, and 0.5% SDS. Hybridization was done for 24 hr at 42°C in 6x SSC, l x Denhardt's solution, 110 p.g/ml yeast tRNA, and 0.05% sodium pyrophosphate, using kinased oligonucleotides at 3 x 10s cpm/ml. Filters were washed four times for 15 rain in 6x SSC, 0.05% sodium pyrophosphate and 30 rain in 6 x SSC, 0.05% sodium pyrophosphate at 45°C and exposed overnight wet against X-ray film (X-Omat AR films; Eastman Kodak, Rochester, NY). Filters were wetted in 6x SSC, washed for 30 min each at 50°C and 55°(3, and reexposed. Sixty-two positive colonies were identified with oligonucleotide I in the first screening; 8 of these clones also hybridized with oligonucleotide 2. E. coil harboring the positive clones, and negative clones as controis, were induced to produce fusion protein by incubation for 2 hr at 42°C. Lysatas were prepared and analyzed by SDS-PAGE and immu-

noblotting with M3A5. One of the clones, clone 70, produced a fusion protein of 205 kd positive with M3A5. Cross-hybridization analysis of all positive (and some negative) clones was performed under high stringency conditions. The clone 70 insert was cut out with BamHI, isolated, and 32p-labelod using the Random-Primed Labelling Kit (Boehringer, Mannheim, FRG) and [a-32p]ATP. Filters treated as described above were hybridized with the probe in 6x SSC, 5x Denhardt's solution, 0.10/0SDS, 10 mM EDTA, 100 i~g/ml herring sperm DNA at 68°C for 12 hr. Filters were washed in 2x SSC, 0.1% SDS at room temperature followed by two washes in 0.1x SSC, 0.1% SDS at 68°C for 30 rain, air dried, and exposed to X-ray film.

DNA Subcloning and Sequencing DNA isolation from transformed bacteria and recombinant DNA manipulations were done using standard procedures (Sambrook et al., 1989). Restriction enzymes and other molecular biology reagents were purchased from Boehringer (Mannheim, FRG) unless stated otherwise. [a-32p]ATP, [y-32P]ATP, and [a-3SS]thio-ATP were obtained from Amersham (Braunschweig, FRG). The inserts of the eight clones were subcloned into plasmid pGEM3Z (Promega). Unidirectional deletion clones of clone 70 were made in both orientations with exonuclease III, using the Nested Deletion Kit of Pharmacia LKB. Dideoxy sequencing of both strands was carried out, using miniprep plasmid DNA and the T7 Sequencing Kit (Pharmacia LKB). The other clones were partially sequenced to test alignment with the sequence of clone 70. Four clones had smaller inserts than clone 70. Two clones, 20 and 64, extended clone 70 to the 5' end. Clone 81 extended clone 70 to the 3' end. The sequences of the clones were identical in all regions of overlap. All clones had an internal BamHI restriction site, which was used to align them. A full-length 13-COPcDNA was constructed in pGEM4Z (Promega) from two restriction fragments of clones 20 and 81 (see Figure 2a), using this BamHI site (nucleotidas 1467-1472 in Figure 21o).To fill the remaining gaps in the sequencing of both strands, synthetic oligonucleotides were used as sequencing primers. Sequence data were compiled and analyzed using the UWGCG package (Devereux et al., 1984). The molecular weight and amino acid composition of ~-COP were analyzed using PEPTIDESORT. In Vitro Transcription/Translation The full-length cDNA was linearized at a 3' restriction site, and G-COP mRNA was produced by in vitro transcription using T7 polymerese (Sambrook et aL, 1989). Translation was done in a rabbit reticulocyte lysate (Promega) in the presence of 20 p.M amino acids (lacking rnethionine) and 0.2 p.Ci/p.I [3SS]methionine (Arnersham, Braunschweig, FRG) for 60 min at 3"PC (for details see Sambrook et al., 1989). Translation products were analyzed by SDS-PAGE and fluorography (Scheel et al., 1990). Northern Analysis The insert of clone 70 was 32p-labeled using the Random-Primed Labelling Kit (Boehringer, Mannheim, FRG) as a hybridization probe. For Northern blot analysis, about 4 p.g of poly(A)+ RNA prepared from rat liver and HeLa cells was electrophoresed through a 1.0% agarose gel in 6.7% formaldehyde, 20 mM MOPS, 5 mM sodium acetate, 0.5 mM EDTA (pH 7.0). RNA was transferred to nitrocellulose filters and hybridized with the 32polabeled probe for 20 hr in 50°/0 formamide, 5x SSC, 5x Denhardt's solution, 0.1% SDS, 100 p.g/ml herring sperm DNA at 42°C. Two final washes were performed in 0.1x SSC, 0.1% SDS at 60°C for 45 rain. The filter was exposed at -70°C with an intensifying screen overnight.

Computer Sequence Analysis TFASTA (Pearson and Lipman, 1988) was used to search the GenEmbl nucleotide data library for any credible homologies with 13-COR The protein data bases combined contain nearly 18,000 nonredundant sequences. The TFASTA technique searches large data bases quickly but is generally not suited to detect extensive distant relationships, relying primarily on residue identity as a scoring criterion. Thus a sensitive sequence comparison technique (Argos, 1987; Rschid et al., 1989) was employed to test and extend, if possible, alignments suggested by the TFASTA procedure. The sensitive technique is based upon residue physical characteristics such as hydrophobicity, the Day-

Cell 662

hoff exchange matrix (Dayhoff et al., 1983), which provides relative weights for the preference of particular amino acid substitutions in proteins, the use of multiple oligopeptids fragment lengths in comparing the two Sequences, and special search plots over the entire probe lengths that allow user identification of the alignment path independent of gap penalty choice. Sequences were multiply aligned with the technique of Vingron and Argos (1989), which first aligns very related sequences and then matches the resulting sequence sets with each other through profile fitting (Gribskov et al., 1987) coupled to a scoring scheme based on the Dayhoff exchange matrix. All pairwise alignments were assessed for significance by the method of Rechid et al. (1989). A mean correlation coefficient was calculated over five amino acid physicochemical characteristics important in protein folding and only over the matched positions in the sequence alignment to be tested. Similar average correlations were determined for sequences matched by the sensitive technique that are known to have unrelated tertiary structures and thus provide a wellfounded baseline for significance. In this way protein sequence biases that are opaque to only random shuffling of the sequences can be detected. Thus a significance of 2.0 standard deviations (a) can be taken with 95% confidence, 3.0 o with 99% confidence, and so forth. Potential sites for posttranslational modifications were identified by search in the PROSITE library (Bairoch, 1989). Antibodies A MAb against VSV-G, P5D4, has been described (Kreis, 1986). In some immunoelectron microscopy experiments an antiserum directed against clathrin light chains (S. Meresse and B. Hoflack, unpublished data) was used. Antibodies against six synthetic peptides of the B-COP sequence were prepared (see underlined regions in Figure 2b): =MDSE" (amino acids 16-29), =KOLQ" (amino acids 107-120), "EAGE" (amino acids 496--513; designated anti-EAGE in the text), =KESE" (amino acids 656-669), =KLVE" (amino acids 767-786), and =LGDIC' (amino acids 940-953). Rabbits were immunized with peptides coupled to keyhole limpet hemocyanin, and antibodies were affinity purified on the peptides coupled to CNBr-Sepharose beads (Pharmacia LKB) as described (Kreis, 1986). All antibodies were reactive with B-COP in immunoblotting. Antibodies anti-EAGE, =KESE," "KLVE" and "LGDK" specifically stained the Golgi complex on methanol/acetonefixed Vero cells. Anti-EAGE also labeled ~-COP on the Golgi complex of paraformaldehyde-fixed cells; two peptide antibodies (=MDSE:' "KDLQ") were negative in immunofluorescence.

Sucrose Gradient Centrlfugation and Gel Filtration Veto cells from eight confluent 10 cm dishes were washed three times with buffer A, scraped into 5 ml of buffer A, and homogenized by forcing the cell suspension ten times through a 25 gauge needle. A 125,000 x g supernatant, obtained after 1 hr centrifugation in a TLA-45 rotor in a Beckman tabletop centrifuge, was concentrated by vacuum dialysis to 800 p.I (,~1.2 mg of protein). Soluble rat liver cytosolic protein (2 mg) was prepared as described above. These samples were loaded on top of 5%-30% (wt/vol) continuous sucrose gradients (11 ml each) made in buffer A and centrifuged in an SW40 rotor at 35,000 rpm for 18 hr at 4°C. Fractions (600 ILl)were collected from the top, and proteins were precipitated with 10o/o trichloroacetic acid (see Duden and Franks, 1988). Samples were analyzed by SDS-PAGE and immunoblotting with M3A5 (Allan and Kreis, 1986). Total protein in the fractions was determined as absorption at 595 nm with the Bio-Rad protein assay (Bio-Rad, Munich, FRG) using bovine serum albumin as a standard. Gel filtration of soluble proteins from rat liver was performed in buffer A at 4°C with a flow rate of 0.2 ml/min using a Superose 6 column and the FPLC system of Pharmacia LKB. Fractions (400 p.I) were collected and processed as described for sucrose gradients. The following reference proteins were used for Mr calibration: thyroglobulin (670,000), apoferritin (440,000), ~-amylase (200,000), alcohol dehydrogenase (150,000), and carboanhydrase (29,000), all from Pharmacia LKB, and IgM (,-~900,000).

Immunofluorescence, Confocal Fluorescence Microscopy, and Immunoelectron Microscopy

Cell Culture, Virus Infection, and Brefeldin A Treatment Vero cells (African green monkey kidney cells, ATCC CCL 81) were maintained and infected with tsO45-VSV (Indiana serotype) as described earlier (Kreis, 1986). In some experiments brafeldin A was added to the cell culture medium at a concentration of 5 i~g/ml. A stock solution of brefeldin A (2 mg/ml in ethanol; Epicentre Technologies, Madison, Wisconsin) was stored at -20°C.

Vero cells grown on glass coverslips were fixed in methanol and acetone at -20°(3 for 4 rain each. Cells were mounted in Mowiol, and epifluorescence microscopy was performed on a Zeiss Axiophot microscope with a 63x, 1.4 oil immersion objective. Photographs were taken on Tmax 3200 negative film (Kodak). The Modular Confocal Microscope (MCM), developed and constructed at EMBL (Rosa et al., 1989), was used for confocal fluorescence microscopy. Cells grown on coverslips ware fixed with 3% paraformaldehyde in PBS for 20 min at room temperature, permeabilized with 0.1% Triton X-100, incubated with antibodies (Kreis, 1986), and mounted in 50% glycerol in PBS. Rhodamine was excited with the 514.5 nm line of an argon ion laser. Series of confocal images of cells were recorded at 0.4 ~m vertical steps. Projections and stereo pairs of each series were calculated. Photographs were taken on Kodak Tmax 100 negative film, using a Polaroid Freeze Frame Recorder. For cryoelectron microscopy, tissue culture cells were fixed with 3% paraformaldehyde in 200 mM HEPES (pH 7.4). An anesthetized rat was perfused with the same fixative to obtain a fixed liver. Fractions enriched in Golgi stacks from rat liver were sadimented by centrifugation at 10,000 x g for 15 min and fixed as described above for cells. Processing of the samples for cryosactioning and labeling with antibodies, 5 nm and 9 nm gold-protein A were performed as described (Griffiths et al., 1984).

Preparation of Stacked Golgi Membranes

Electrophoresis and Immunoblottlng

The livers (16 g) of.two rats (Sprague-Dawley) were removed and immediately placed into 25 ml of ice-cold buffer A (100 mM KPO4, 5 mM MgCI2 [pH 6.7]) containing 0.5 M sucrose. The tissue was first minced and then forced through a stainless steel mesh (pore size 0.5 ram) with a porcelain pestle. Six milliliters of homogenate was overlaid over six sucrose step gradients each in SW40 tubes (4 ml of buffer A containing 0.86 M sucrose, underlaid with buffer A containing 1.3 M sucrose). Finally, 2.5 ml of buffer A containing 0.25 M sucrose was overlaid. Gradients were centrifuged in an SW40 rotor at 29,000 rpm for 1 hr at 4°C. Golgi stacks that accumulated at the 05M/0.86M sucrose interphase were collected in a total volume of 5 ml with a Pasteur pipette. These fractions were diluted to 0.25 M sucrose with buffer A. Aliquots of 1 ml were used in the experiments. A control sample was incubated on ice with no additions. To two other aliquots, an ATP-regenerating system containing 10 mM creatine phosphate, 2.5 mM Na-ATP, and 100 lig/ml creatine phosphokinase was added, with or without addition of 20 I~M GTPTS. Golgi stacks were recovered after incubation for 15 min in a water bath at 3"/°C by centrifugation at 10,000 x g for 15 min at 4°(3 and processed for electron microscopy as described below.

Reduced proteins were separated by SDS-PAGE (Scheel et al., 1990). For the analysis of in vitro translation products, gels were stained with Coomassie blue, destained, washed in water for 30 ntin, incubated in 1 M salicylate for 20 min, and vacuum dried. Fluorographs on X-ray film (X-Omat-S films; Eastman Kodak, Rochester, NY) were taken at -70°C (see Scheel et al., 1990). Proteins separated by SDS-PAGE were transferred onto nitrocellulose filters for immunoblotting. The filters were incubated with antibodies and labeled bands visualized as described (Allan and Kreis, 1986).

Acknowledgments We are grateful to Dr. Keith Stanley for providing the rat liver cDNA library. We thank Heinrich Gausepohl for synthesis of peptides, and Armin Bosserhoff and Heinz Horstmann for skillful technical assistance with protein sequencing and immunoalectron microscopy, respectively. We also thank Drs. Stephane Meresse and Bernard Hoflack for giving us the antiserum against clathrin light chains, Marino Zerial for help with the cloning of B-COR Thomas Kirchhausen and Margaret

110 kd Adaptin Homolog on Non-Clathrin-Coated Vesicles 663

Robinson for sharing sequences of adaptins prior to publication, as well as Felix Wieland and James Rothman for sharing unpublished information on rabbit liver B-COP. Martin Vingron and Dr. Stephen Fuller helped with sequence comparisons, and Andreas Merdes with confocal microscopy; Drs. James Pryde and Graham Warren provided us with the protocol for the preparation of stacked Golgi cisternae. We acknowledge many helpful and stimulating discussions with Dr. Richard Klausner and our colleagues in the EMBL Cell Biology Program, and Drs. Ed Hurt and Kai Simons for critically reading the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked =advertisement" in accordance with 18 USC Section 1734 solely to indicate this fact. Received December 10, 1990; revised January 10, 1991. References

Ahle, S., and Ungewickell, E. (1989). Identification of a clathrin binding subunit in the HA2 adaptor protein complex. J. Biol. Chem. 264, 20089-20093. Ahle, S., Mann, A., Eichelsbacher, U., and Ungewickell, E. (1988). Structural relationships between clathrin assembly proteins from the Golgi and the plasma membrane. EMBO J. 7, 919-929. Aizawa, H., Emori, Y., Murofushi, H., Kawasaki, H., Sakai, H., and Suzuki, K. (1990). Molecular cloning of a ubiquitously distributed microtubule-associated protein with Mr 190,000. J. Biol. Chem. 265, 13849-13855. Allan, V. J., and Kreis, T. E. (1986). A microtubule-binding protein associated with membranes of the Golgi apparatus. J. Cell Biol. 103, 2229-2239. Allan, V. J., Karsenti, E., and Kreis, T. E. (1988). Cell cycle-dependent phosphorylation of a Golgi-associated microtubule-binding protein. J. Cell Biol. 107, 759a. Argos, P. (1967). A sensitive procedure to compare amino acid sequences. J. Mol. Biol. 193, 385-396. Bairoch, A. (1989). Prosite: a dictionary of protein sites and patterns, 4th ed., October 1989, Dept. de Biochemie M(~dicale, CMU, Universit~ de G6n~ve, Geneva, Switzerland. Bressan, G. M., and Stanley, K. K. (1967). pUEX, a bacterial expression vector related to pEX with universal host specificity. Nucl. Acids Res. 15, 10056. Brodsky, F. (1988). Living with clathrin: its role in intracellular membrane traffic. Science 242, 1396-1402. Cooper, M. S., CornelI-Bell, A. H., Chernjavsky, A., Dani, J. W., and Smith, S.J. (1990). Tubulovesicular processes emerge from transGolgi cisternae, extend along microtubules, and interlink adjacent trans°Golgi elements into a reticulum. Cell 61, 135-145. Dayhoff, M. O., Barker, W. C., and Hunt, L. T. (1983). Establishing homologies in protein sequences. Meth. Enzymol. 91, 524-545. Devereux, J., Haeberli, P., and Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucl. Acids Res. 12, 367-395. Doms, R. W., Russ, G., and Yewdell, J. W. (1989). Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum. J. Cell Biol. 109, 61-72. Donaldson, J. G., Lippincott-Schwartz, J., Bloom, G. S., Kreis, 11 E., and Klausner, R. D. (1990). Dissociation of a 110kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A action. J. Cell Biol. 111, 2295-2306. Donaldson, J. G., Lippincott-Schwartz, J., and Klausner, R. D. (1991). Guanine nucleotides modulate the effects of brefeldin A in semipermeable cells: regulation of the association of a 110kD peripheral membrane protein with the Golgi apparatus. J. Cell Biol., in press. Duden, R., and Franks, W. W. (1988). Organization of desmosomal plaque proteins in cells growing at low calcium concentrations. J. Cell Biol. 107, 1049-1063. Farquhar, M. G. (1985). Progress in unraveling pathways of Golgi traffic. Annu. Rev. Cell Biol. 1, 447-488.

Fujiwara, T., Oda, K., Yokota, S., Takatsuki, A., and Ikehara, Y. (1988). Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum. J. Biol. Chem. 263, 18545-18552. Gausepohl, H., Trosin, M., and Frank, R. (1986). An improved gasphase sequenator including on-line identification of PTH amino acids. In Advanced Methods in Protein Microsequence Analysis, B. Wittmann-Liebold, J. Salnikow, and V.A. Erdmann, eds. (Berlin: Springer Verlag), pp. 149-160. Glickman, J. N., Conibear, E., and Pearse, B. M. F. (1989). Specificity of binding of clathrin adaptors to signals on the mannose-6phosphate/insulin-like growth factor II receptor. EMBO J. 8, 1041-1047. Gribskov, M., McLachlan, A. D., and Eisenberg, D. (1987). Profile analysis: detection of distantly related proteins. Proc. Natl. Acad. Sci. USA 84, 4355-4358. Griffiths, G., and Simons, K. (1986). The trans Golgi network: sorting at the exit site of the Golgi complex. Science 234, 438-443. Griffiths, G., McDowall, A., Back, R., and Dubochet, J. (1984). On the preparation of cryosections for immunocytochemistry. J. Ultrastruct. Res. 89, 65-78. Griffiths, G., Fuller, S. D., Back, R., Hollinshead, M., Pfeiffer, S., and Simons, K. (1989). The dynamic nature of the Golgi complex. J. Cell Biol. 108, 277-297. Ho, W. C., Allan, V. J., van Meer, G., Berger, E. G., and Kreis, T. E. (1989). Reclustering of scattered Golgi elements occurs along microtubules. Eur. J. Cell BioK 48, 250-263. Ho, W. C., Storrie, B., Pepperkok, R., Ansorge, W., Karecla, P., and Kreis, T. E. (1990). Movement of interphase Golgi apparatus in fused mammalian cells and its relationship to cytoskeletal elements and rearrangement of nuclei. Eur. J. Cell Biol. 52, 315-327. Karrenbauer, A., Jeckel, D., Just, W., Birk, R., Schmidt, R. R., Rothman, J. E., and Wieland, F. T. (1990). The rate of bulk flow from the Golgi to the plasma membrane. Cell 63, 259-267. Keen, J. H. (1990). Clathrin and associated assembly and disassembly proteins. Annu. Rev. Biochem. 59, 415-438. Keen, J. H., and Beck, K.A. (1989). Identification of the clathrinbinding domain of assembly protein AP2. Biochem. Biophys. Res. Commun. 158, 17-23. Keen, J. H., Chestnut, M. H., and Beck, K. A. (1967). The clathrin coat assembly polypeptide complex. J. Biol. Chem. 262, 3864-3871. Kirchhausen, T. (1990). Identification of a putative yeast homolog of the mammalian ~ chains of the clathrin-associated protein complexes. Mol. Cell. Biol. 10, 6089-6090. Kirchhausen, T., Nathanson, K. L., Matsui, W., Vaisberg, A., Chow, E. R, Burne, C., Keen, J. H., and Davis, A. E. (1989). Structural and functional division into two domains of the large (100- to 115okDa) chains of the clathrin-associated protein complex AP-2. Proc. Natl. Acad. Sci. USA 86, 2612-2616. Kornfeld, R., and Kornfeld, S. (1985). Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 54, 631-664. Kozak, M. (1967). An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucl. Acids Res. 15, 8125-8143. Kreis, T. E. (1986). Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. EMBO J. 5, 931-941. Kreis, T. E. (1990). Role of microtubules in the organization of the Golgi apparatus. Cell Motil. Cytoskel. 15, 67-70. Lee, C., Levin, A., and Branton, D. (1967). Copper staining: a five minute protein stain for sodium dodecyl-sulfate-polyacrylamide gels. Anal. Biochem. 166, 308-312. Leelavathi, D. E., Estes, L. W., Feingold, D. S., and Lombardi, P. (1970). Isolation of a Golgi-rich fraction from rat liver. Biochim. Biophys. Acta 211, 124-138. Lewis, S.A., Wang, D., and Cowan, N.J. (1988). Microtubuleassociated protein MAP-2 shares a microtubule binding motif with tau protein. Science 242, 936-939. Lindsay, J. D., and Ellisman, M. H. (1985). The neuronal endomem-

Cell 664

brana system. I1. The multiple forms of the Golgi apparatus cis element. J. Neurosci. 5, 3124-3134. Lippincott-Schwartz, J., Yuan, L. C., Bonifacino, J. S., and Klausner, R. D. (1989). Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell 56, 801-813. Lippincott-Schwartz, J., Donaldson, J.G., Schwaizer, A., Berger, E. G., Hauri, H.-R, Yuan, L. C., and Klausner, R. D. (1990). Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell 60, 821-836. Lucocq, J. M., Berger, E. G., and Warren, G. (1989). Mitotic Golgi fragments in HeLa cells and their role in the reassembly pathway. J. Cell Biol. 109, 463-474. Luzio, J. R, Brake, B., Bating, G., Howell, K. E., Braghetta, R, and Stanley, K. K. (1990). Identification, sequencing and expression of an integral membrane protein of the trens-Golgi network (TGN38). Biochem. J. 270, 97-102. Mahaffey, D. T., Peeler, J. S., Brodsky, F. M., and Anderson, R. G. W. (1990). Clathrin-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity. J. Biol. Chem. 265, 16514-16520. Malhotra, V., Serafini, T., Orci, L., Shepherd, J. C., and Rothman, J. E. (1989). Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack. Cell 58, 329-336. Marlin, K. S., and Simons, K. (1983). Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation. Cell 34, 233-243. Melancon, R, Glick, B. S., Malhotra, V., Weidman, P. J., Serafini, T., Gleason, M. L., Orci, L., and Rothman, J. E. (1997). Involvement of GTP-binding "G" proteins in transport through the Golgi stack. Cell 51, 1053-1062. Misumi, Y., Miki, K., Takatsuki, A., Tamura, G., and Ikehara, Y. (1986). Novel blockade by brsfeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes. J. Biol. Chem. 261, 11398-11403. Morsno, S., and Nurse, R (1990). Substrates for p34CdC2: in vivo veritas? Cell 61, 549-551. Morris, S. A., Ahle, S., and Ungewickell, E. (1989). Clathrinocoated vesicles. Curr. Opinion Cell Biol. 1, 684-690. Noble, M., Lewis, S. A., and Cowan, N. J. (1989). The microtubule binding domain of microtubule-associated protein MAP1B contains a repeated sequence motif unrelated to that of MAP2 and tau. J. Cell Biol. 109, 3367-3376. Novick, R, Field, C., and Schekman, R. (1980). Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21, 205-215. Obar, R. A., Collins, C. A., Hammarback, J. A., Shpetner, H. S., and Vallee, R. B. (1990). Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of GTP-binding proteins. Nature 347, 256-261. Orci, L., Ravazzola, M., Amherdt, M., Louvard, D., and Perrelet, A. (1985). Clathrin-immunoreactive sites in the Golgi apparatus are concentrated at the trans pole in polypeptide hormone-secreting cells. Proc. Natl. Acad. Sci. USA 82, 5385-5389. Orci, L., Glick, B. S., and Rothman, J. E. (1986). A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack. Cell 46, 171-184. Orci, L., Malhotra, V., Amherdt, M., Serafini, T., and Rothman, J. E. (1989). Dissection of a single round of vesicular transport: sequential intermediates for intercisternal movement in the Golgi stack. Cell 56, 357-368. Palade, G. E. (1975). Intracellular aspects of the process of protein synthesis. Science 189, 347-358. Pearse, B.M.F. (1988). Receptors compete for adaptors found in plasma membrane coated pits. EMBO J. 7, 3331-3336. Pearse, B. M. F., and Bretscher, M. S. (1981). Membrane recycling by coated vesicles. Annu. Rev. Biochem. 50, 85-101.

Pearse, B. M. F., and Robinson, M. S. (1984). Purification and properties of 100-kd proteins from coated vesicles and their reconstitution with ciathrin. EMBO J. 3, 1951-1957. Pearson, W. R., and Lipman, D. J. (1988). Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444-2448. Pelham, H. R. B. (1989). Control of protein exit from the endoplasmic reticulum. Annu. Rev. Cell Biol. 5, 1-23. Pfeffer, S. R., and Rothman, J. E. (1987). Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu. Rev. Biochem. 56, 829-852. Ponnambalam, S., Robinson, M. A., Jackson, A. P., Peiperl, L., and Parham, R (1990). Conservation and diversity in families of coated ves!cle adaptins. J. Biol. Chem. 265, 4814-4820. Rambourg, A., and Clermont, Y. (1990). Three-dimensional electron microscopy: structure of the Golgi apparatus. Eur. J. Cell Biol. 51, 189-200. Rechid, R., Vingron, M., and Argos, R (1989). A new interactive protein sequence alignment program and comparison of its results with widely used algorithms. CABIOS 5, 107-113. Robinson, M. S. (1987). 100kD coated vesicle proteins: molecular heterogeneity and intracellular distribution studied with monoclonal antibodies. J. Cell Biol. 104, 887--895. Robinson, M. S. (1989). Cloning of cDNAs encoding two related 100kD coated vesicle proteins (a-adaptins). J. Cell Biol. 108, 833-842. Robinson, M. S. (1990). Cloning and expression of y-adaptin, a component of clathrin-coated vesicles associated with the Golgi apparatus. J. Cell Biol. 111, 2319-2326. Rosa, R, Weiss, U., Pepperkok, R., Ansorge, W., Niehrs, C., Stelzer, E. H. K., and Huttner, W. B. (1989). An antibody against secretogranin I (chromogranin B) is packaged into secretory granules. J. Cell Biol. 109, 17-34. Rossmann, M. G. (1987). The evolution of RNA viruses. Bioessays 7, 99-103. Rossmann, M. G., and Argos, P. (1981). Protein folding. Annu. Rev. Biochem. 50, 497-532. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory). Saraste, J., and Kuismanen, E. (1984). Pre- and post-Golgi vacuoles operate in the transport of Semliki forest virus membrane glycoproteins to the cell surface. Cell 38, 535-549. Scheel, J., Matteoni, R., Ludwig, T., Hoflack, B., and Krais, T. E. (1990). Microtubule depolymerization inhibits transport of cathepsin D from the Golgi apparatus to lysosomes. J. Cell Sci. 96, 711-720. Schweizer, A., Fransen, J. A. M., Matter, K., Krais, T. E., Ginsel, L., and Hauri, H.-P. (1990). Identification of an intermediate compartment involved in protein transport from the endoplasmic reticulum to Golgi apparatus. Eur. J. Cell Biol. 53, 185-196. Serafini, T., Stenbeck, G., Brecht, A., Lottspeich, F., Orci, L., Rothman, J. E., and Wialand, F. T. (1991). Identification of ~-COP, a subunit of the coat of Golgi-derived (non-clathrin) coated vesicles, as a homologue of the clathrin-coated vesicle coat protein ~-adaptin: N,atura, in press. Siegel, L. M., and Monty, K.J. (1966). Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hYdroxylamine reductases. Biochim. Biophys. Acta 112, 346-362. Tanford, C. (1961). Physical Chemistry of Macromolecules (New York: John Wiley & Sons), pp. 356-361. Tooze, J., and Tooze, S. A. (1986). Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells. J. Cell Biol. 103, 839-850. Vingron, M., and Argos, R (1989). A multiple sequence alignment algorithm with improved sensitivity. CABIOS 5, 115-121. Wieland, F.T., Gleason, M. L., Serafini, T. A., and Rothman, J. E. (1987). The rate of bulk flow from the endoplasmic reticulum to the cell surface. Cell 50, 289-300.

110 kd Adaptin Homolog on Non-Clathrin-Coated Vesicles 665

Yang, J. T., Laymon, R. A., and Goldstein, L. S. B. (1989). A threedomain structure of kinesin heavy chain revealed by DNA sequence and microtubule binding analyses. Cell 56, 879-889. GenBank Accession Number

The accession number for the sequence reported in this paper is X57228.