Two rat homologs of clathrin-associated adaptor proteins

Gene, 146 (1994) 279-283 0 1994 Elsevier Science B.V. All rights reserved.

GENE

279

0378-1119/94/$07.00

08052

Two rat homologs of clathrin-associated (Assembly

Jonathan

proteins;

membrane

trafficking;

recycling;

Pevsner, Walter Volknandt”,

adaptor proteins

vesicles; ray electric lobe)

Brian R. Wong and Richard H. Scheller

Department qf Molecular and Cellular Physiology, Howard Hughes Medical Institute, Beckman Center, Stanford University Medical School, Stanford, CA 94305, USA Received A.D. Riggs: 14 December

1993; Revised/Accepted:

15 March/22

March

1994; Received at publishers:

25 April 1994

SUMMARY

A cDNA clone predicted to encode a 46 757-Da protein was isolated from a library derived from the electric lobe of the ray Discopyge ommata. Two rat homologs, p47A and p47B, were subsequently isolated. These three proteins share approx. 80% amino acid (aa) identity to each other and have 27-30% aa identity to rat AP50 and mouse AP47, the medium-chain subunits of adaptor complexes associated with clathrin-coated vesicles. These complexes are involved in receptor-mediated pathways of intracellular transport. Rat p47A mRNA is expressed in all tissues examined, including brain, heart, kidney, liver, lung, muscle and spinal cord. Rat p47B mRNA is detected exclusively in brain and spinal cord, and may participate in nervous system-specific functions such as biogenesis or recycling of synaptic vesicles.

INTRODUCTION

Vesicles mediate the selective transport of a variety of proteins within the secretory pathway. A set of coat proteins attach to vesicles as they bud from donor organelles (Hurtley, 1991). These vesicles are either clathrin-coated or, in the case of vesicles responsible for intra-Golgi transport, they are non-clathrin coated (Waters et al., 1991). Two major classes of clathrin-coated vesicles have been elucidated (Ahle et al., 1988; Pearse and Robinson, 1990; Keen, 1990; Schmid, 1992): those which internalize plasma membrane receptors in receptor-mediated endocytosis,

and those

associated

with the Golgi

apparatus

Correspondence to: Dr. R.H. Scheller, Howard Hughes Medical Institute, Stanford U. Medical Center, Stanford, CA 94305, USA. Tel. (1-415) 723-9075; Fax (1-415) 725-4436. *Current address: AK Neurochemie, Zoologisches Institut der J.W. Goethe-Universitlt,

W-6000

Frankfurt,

Germany.

Tel. (49-69)

798-4829. Abbreviations:

aa,

amino

acid(s);

AP,

assembly

adaptor; bp, base pair(s); D., Discopyge; kb, kilobase nucleotide(s); ORF, open reading frame. s

polypeptide

or

or 1000 bp; nt,

that transport newly synthesized proteins to lysosomes or participate in the formation of regulated secretory granules. Each type of clathrin-coated vesicle relies on a unique heterotetrameric complex of proteins called adaptors (Pearse, 1985) or assembly polypeptides (Zaremba and Keen, 1983). These complexes promote clathrin cage formation (Keen et al., 1979; Zaremba and Keen, 1983), and also specifically bind the cytoplasmic tails of membrane proteins and direct their transport. Adaptor complexes have lOO-llO-kDa subunits called adaptins, a medium chain of 50 kDa or 47 kDa, and a small chain of 17-19 kDa. While only two adaptor complexes have been well characterized, dozens of proteins are transported via coated vesicles. These proteins include cell-surface neurotransmitter receptors, growth factor receptors, lysosomal enzymes, immunoglobulins and recycling synaptic vesicle proteins. We report the isolation and sequence analysis of a cDNA encoding a novel 47-kDa protein (~47) from the electric ray Discopyge ommata that is homologous to the medium chain of adaptor complexes. We have also isolated two rat homologs that may be constituents of novel adaptor complexes.

EXPERIMENTAL

While

AND DISCUSSION

AP50

has been proposed

as an autokinase

Keen et al., 1987; Myers and Forgac, proteins

(a) Identification and sequence analysis of three p47 cDNA clones

The p47 proteins

lmmunoscreening pression

of 250000

library

derived

ray D. ommata resulted

positive

1.8-kb clone. Analysis

cDNA

clone

encode

a protein

revealed

the electric

library

long

clones.

ORF

clone,

sequences,

hybridized

of a of the

predicted

to

we screened

plaques

Of these, 21 (denoted

denoted

of the

a

we identified rat p47A) be-

rat p47B,

only

analysis.

weakly

cross-

with p47A and was used to isolate a full-length

clone from a rat cerebral

at aa positions disulfide

bonds

the proteins, complex

to known

contain

1993), the three p47

kinases (Hunter,

four conserved

29. 97. 209 and important

236. These

in the secondary

or intrasubunit

bonds

1987).

Cys residues may form structure

of

in a multimolecular

(see below). The Cys236 residue is also conserved

in AP50, AP47 and YAP54.

ray ~47. To charac-

longed to a single class based on DNA sequence Another

ex-

(c) Regional localization The regional

using the ray p47 clone as

From a screen of 400000

22 positive

lobe

of the nt sequence

a single

mammalian

cord cDNA

of a cDNA

in the identification

of 418 aa, denoted

terize the related a probe.

plaques

from

electric

rat spinal

lack homology

(e.g..

cortex cDNA

library.

of messenger RNA

expression

of p47A and p47B was investi-

gated by RNA blot analysis. in all tissues examined

The p47A clone is expressed

(brain,

heart, kidney,

muscle and spinal cord) with an apparent of 2.4 kb, corresponding

approximately

liver, lung,

transcript

size

to the size of the

cDNA (Fig. 2A). In contrast, the p47B clone is expressed almost exclusively in brain with lower amounts in spinal

The ray p47 clone ( 1773 nt; GenBank accession No. LO7072), rat p47A (2186 nt; accession No. LO7073) and rat p47B (3306 nt; accession No. LO7074) each contains an ORF of 1254 bp. The start Met’s are encoded by nt 27-29, 44-46 and 32-34, respectively.

cord (Fig. 2B). The transcript size of p47B RNA was approx. 3.1 kb, which is comparable to the size of the cDNA (3.3 kb). Equal amounts of RNA were loaded on the gels as indicated by methylene blue staining (Fig. 2C). The lower molecular weight bands at about 1.6 kb (Fig. 2A; Fig. 2B lanes 6 and 7) may represent alternative

(h) Deduced aa sequences and homologies Each of the three p47 clones is predicted to encode a 418-aa protein of of 46757 Da (ray p47), 46980 (rat p47A) or 46873 (rat p47B). A database search with the FASTA program revealed significant homology between these proteins and mouse AP47, rat AP50 and yeast YAP54 (Thurieau et al., 1988; Nakayama et al., 1991) (Table I and Fig. 1). The three p47 proteins form a distinct subgroup, sharing 79+84% aa identity with no gaps in the alignment. The three proteins share 89+91% aa

transcripts

similarity (Table I). Each of these proteins shares statistically significant aa identity (27-30% identity) to AP47, AP50 and YAP54. Computer database searches revealed no significant homologies of the three p47 proteins with entries in the SwissProt database other than the three medium chains.

TABLE

or degradation

products.

(d) Conclusions (I) In this paper we report the identification of three highly related homologs of clathrin-coated vesicleassociated adaptor complex medium chains. The cDNA clone encoding ray p47 was used to isolate two rat clones encoding p47A and p47B. While rat p47A mRNA is expressed in all tissues examined, p47B is predominantly brain-specific. The major significance of these findings is that the medium chains we have isolated may be constituents of novel adaptor protein complexes. At present two distinct types of clathrin-coated vesicle associated adaptors have been identified (Keen, 1990; Pearse and Robinson, 1990). HA-l, which is localized primarily to the Golgi apparatus, binds the cytoplasmic tail of the

I

Comparison

of protein

homologies

Protein

ray p47

ray p47 rat p47A rat p47B AP50 AP47 YAP54

100%

I’dt

p47A

82% (89,[0] 100%

rat p47B 79% (90)[0] 84% (91)[0] 100%

a Percent aa identities and aa similarities (in parentheses) were calculated according WI, USA). The number of gaps in the alignment is indicated in brackets.

AP50 28% (54)[ 1 I] 27% (54)[ lo] 29% (55)[11] 100%

to the Bestfit program

AP47

YAP54

28% (55)[9]

27% (53)[ 131

29% (54)[S] 30% (56)[8] 40% (63)[5] 100%

from Genetics

27% 27% 38% 57% 100% Computer

(54)[ 121 (50)[ IO] (SS)[S] (74)[8]

Group

(Madison,

281

TNIARTSFFH LAHGGVRFMW LNHNGLEYLF

VKRSN1WL.A IKHNNLY LVA IOHNDLYVVA

L

AVTKQNVNAA TSKKN. ACVS IVTSLSANAA

MVFQFLYKMC LVFSFCYKVV AIFTFLHKLV

DVMAAYFGK OVFSEYF’KEL EVLSDYLKTV

Nf;‘FPLATES: D.FGYP0NSE.T DFGYPQTTDS DYCIPQITET

NiLKELtKPP NX’UEL~KPP N’iiL’KE.Lf..KPP GALKTFIT00 K’I’LQEY JTOE KM~K~YITQK

TEL T t L T.t..L

S:EENI.&NN:FN, E’E:ES I’RDN:F$! E:E’EstRDN.fV

LIY’EUtDEI J.1 v”E_tfDELM ~‘IYE’LLDEvM

HQTKEEOSOI TG....APRP AKKKRNATRP

TSQVTGOIGW PATVTNAVSW F‘VALTNSVSW

RREGIKYRRN RSEGIKYRKN RPEGITHKKN

ELFLDVLESV EVFLDVIEAV EAFLDIVESI

LRSE$I.VGSJK LRSEIIGDVK

MRVFL’SGMPE VNSKLSGIPH

LRLGLNDKVL LKLGIKDKGI

F. FSKYLDDDTN

P

GI

. KS. GH K L SF.KLVKS.

. . .O . E

NLLMSPOGCIV NLLVSANENV NMLMTOKGQV

IPSASATTSD n IJ

.

.

._._.

LDDVS LDDVS s T

1: 1: 1: 1: 16 k&i

FHPC’VRFKRW FHPCIRFKRW F,H.P~VRFK w FHO&LS;F PHOC%RLSRF FttClCYRLSKF

ESE ES? ESE, DSE ENDRTISFIP ENEKIITFIP

I EKHS.HS.RI VOVHSNS.RIE

YMVKAKS~FK IHCKAKAOIK

KGiAbiiSKS DNTGRGKSKS NNTETDKKPS

..______ ITSSSATNKK

. ..VELEDVK KVNIELEDLK

PDGEFE~MsY PDGKFDCMNY

RLNTHVKPLI RL~TTIKPL~

w I. w~.....DvN

RRSSANNVEI RKSfATNVEI

HIPVPNDADS LJPVPDDADT

PKFKTTVGSV PTFKYSHGSL

KWVPENSEIV KYVP’EKSAI

EYLMRAHFGL EYSMSAELGi

PSVEAEDKE. PSISNNEDGN

RTMPKSNAEI

GKPPISVKFE SKGPVClIKkO

VRYLKVFEPK VRYtKII.EK VRYCKINEPK

SG...YOALP LO.. .YKSYP

WVRYITONG. WVRYITQSGD

DYQLRTQ DYTIRLT

ESV

E

L

WSVKSFPGGK WKIRSFPGGK

%PYFTTSG:I’O IPYFTTSGIQ

AP47 YAP54

Fig. 1. Alignment of six proteins: ray ~47, rat p47A, rat p47B, rat AP50 (Thurieau et al., 1988) mouse AP47 and yeast YAP54 (Nakayama et al., 1991). The predicted aa of ray p47 are from a clone isolated by immunoscreening a hgtll cDNA library (prepared by Dr. Fabio Rupp and derived from the electric lobe of the electric ray D. ommata) with an antiserum directed against cholinergic synaptic vesicles of Torpedo marmorata electric organ (Volknandt and Zimmermann, 1990). Immunoscreening by epitope selection was performed as described (Sambrook et al., 1989; Rupp et al., 1991); a second unrelated positive clone was also isolated but had no ORFs and was not further characterized. The rat p47 cDNAs were isolated from an embryonic day 13 spinal cord library (Rupp et al., 1991) and an adult rat cerebral cortex cDNA library (Stratagene). The aa residues of the three p47 proteins are numbered. Aa identities between four or more of the six proteins are shaded; aa identities among the three p47 proteins are boxed. Alignments were determined by the GAP and PILEUP algorithms. Regions of high conservation, with 61% aa identity between at least four of the six proteins, are: A (~47, aa 799134), B (aa 167-187) C (aa 200-220) D (aa 228-263) and E (aa 385-418).

282

mannosc-6-pllospllatc

A

target proteins

1234567

rcccptor

to lysosomes.

tails of many cell-surface rin receptor.

1988), lysosomal the

coated plexes

M--

0.24

have a native and Robinson,

Bazari.

1987; Virshup

(Beltzer

internalization

vesicles followed

compartments.

The HA-I

molecular

mass

1984 Keen, and Bennett,

Spicss.

and HA-2 comof 250~-350 kDa

1987; Manfredi

and

1988).

teins may bc constituents

of adaptor-related adaptins

with the p47 proteins

and

by their transfer

The nature

ray p47 clone was immunoisolated

( Pearsc,

in clathrin-

(2) While it has not been demonstrated, of the putative

the transfcr-

receptor

(Sosa et al., 1993) and

receptor to their

(Pearse

associate

B

leads

pits and coated

to endosomal

including

lipoprotein

acid phosphatase

asialoglycoprotein

1991). This

HA-2 binds the cytoplasmic

rcccptors

the low-density

ct al., 1989) to

(Glickman

the p47 procomplexes.

and small chains that remains

unknown.

from a cDNA

The library

using an antiserum directed against purified synaptic vesicles, and we therefore suggest that this protein may be

1234567 + I-

kb 7.5 4.4

-

2,4

4-

1.4

-a-

0.24

c 1234567

Fig. 2. Northern analysis of (A) rat p47A and (B) rat ~4713 expression. 20 pg of total RNA were isolated (Chirgwin et al., 1979) from rat heart (lane I ), kidney (lane 2), liver (lane 3), lung (lane 4). muscle (lane 5). spinal cord (lane 6) and brain (lane 7), and electrophoresed on a 2.2 M formaldehyde-l % agarose gel. Sampies were transferred to Nytran (Schleicher&Schuell), and probed with random primer lahelled cDNA clones at 65-C for 15 h in 0.2 M Na.pllosphate (pH 7.2)/l mM EDTA(l% bovine serum alburnin~5~~ sodium dodecyl sulfate. RNA markers in kb are indicated by arrowheads. (C) Methylene blue staining of RNA blot probed in (A).

present on a subset of synaptic vesicles or copurifying vesicles. Following the fusion of synaptic vesicles with the plasma membrane and the exocytotic release of neurotransmitter, the vesicle membrane is believed to bc rapidly internalized via coated pits and coated vesicles (Heuser and Reese, 1973; Holtzman, 1992: Maycox et al.. 1992). These vesicles then rapidly uncoat and may fuse with an endosomal compartment. Nascent vesicles may bud from the cndosome via a distinct coated vesicle pathway, and then be reloaded with neurotransmittcr. regenerating the mature vesicles. The antiserum against synaptic vesicles used to isolate ray p47 may have recognized a coat protein still present on at least some fraction of the vesicle membrane. (3) Another set of coat proteins has been identified in non-clathrin-coated vesicles involved in the bulk flow of constitutive protein transport within the Golgi apparatus (Waters et al., 199 1; reviewed in Hurtley, 1991). These cytosolic coat proteins (COPS), including cl-COP (160 kDa), B-COP ( 110 kDa), *{-COP (98 kDa), S-COP (61 kDa), as well as lower molecular mass components (~36, p35 and ~20). form a high molecular mass complex referred to as a coatomer. Several coatolner-associ~~ted proteins of 40-50 kDa are also evident (Waters et al.. 1991; Serafini et al., 199 I ). B-COP is significantly homologous to P-adaptin although the two proteins share only 17% aa identity over 450 aligned aa (Duden et al., I991 ). p47 may be a homolog of a coatomer protein. There is immunological and biochemical evidence for heterogeneity among coated vesicles ( Puszkin et al., 1989) and p47 might also associate with coated vesicles. (it) In conclusion. we have identified three proteins which are potentiai C~~nstitLIents of novel adaptor complexes. A description of their intracellular localization in

283 neurons is likely to elucidate new pathways of membrane trafficking. The analysis of putative protein constituents of the cytosolic brain complex(es) will clarify the relation of the p47 proteins to the adaptor complexes. The neuralspecific medium chain homolog may provide insight into the recycling or targetting processes unique to neurons, such as recycling of the synaptic vesicle membrane in the nerve terminal.

ACKNOWLEDGEMENTS

We thank Dr. Tomas Kirchhausen for sharing unpublished data and N. Varg for manuscript preparation. This work is supported by the National Institute of Mental Health.

REFERENCES Able, S., Mann, A., Eichelsba~her, U. and Ungewickell, E.: Structural relationships between clathrin assembly proteins from the Golgi and the plasma membrane. EMBO J. 7 (1988) 919-929. Beltzer, J.P. and Spiess, M.: In vitro binding of the asialoglycoprotein receptor to the B adaptin of plasma mcmbrne coated vesicles. EMBO J. 10 (1991) 373553742. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J.: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18 (1979) 5294-5299. Duden. R., Griffiths, G., Frank, R., Argos, P. and Kreis, T.E.: &COP, a 110 kd protein associated with non-clathrin-coated vesicles and the Golgi complex, shows homology to S-adaptin. Cell 64 (1991) 649-665. Glickman, J.N., Conibear, E. and Pearse, B.M.F.: Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate~insulinlike growth factor II receptor. EMBO J. 8 (1989) 1041-1047. Heuser, J.E. and Reese, T.S.: Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57 (1973) 315-344. Holtzman, E.: Membrane trafficking in neurons. Curr. Opin. Neurobiol. 2 (1992) 607-612. Hunter, T.: A thousand and one protein kinases. Cell 50 (1987) 823-829. Hurtley, SM.: Clathrin- and non-clathrin-coated vesicle adaptors. Trends Biochem. Sci. 16 (1991) 165-6. Keen, J.H.: Clathrin assembly proteins: affinity purification and a model for coat assembly. 3. Cell Biol. IO5 (1987) 198991998. Keen, J.H.: Ciathrin and associated assembly and disassembly proteins. Annu. Rev. Biochem. 59 (1990) 415-438. Keen, J.H., Chestnut, M.H. and Beck, K.A.: The clathrin coat assembly poiypeptide complex: autophosphorylation and assembly activities. J. Biol. Chem. 262 (1987) 3864-3871. Keen, J.H., Willingham, M.C. and Pastan, I.H.: Clathrin-coated vesicles: isolation, dissociation and factor-dependent reassociation of clathrin baskets. Cell 16 (1979) 303-312.

Manfredi, J.J. and Bazari, W.L.: Purification and chara~te~zation of two distinct complexes of assembly polypeptides from calf brain coated vesicles that differ in their polypeptide composition and kinase activities. J. Biol. Chem. 262 (1987) 12182-12188. Maycox, P.R., Link, E., Reetz, A., Morris, S.A. and Jahn, R.: Clathrincoated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling. J. Cell Biol. 118 (1992) 1379-1388. Myers, M. and Forgac, M.: The coated vesicle vacuolar (H+)-ATPase associates with and is phosphorylated by the 50-kDa polypeptide of the clathrin assembly protein AP-2. J. Biol. Chem. 268 (1993) 9184-9186. Nakayama, Y., Goebl, M., O’Brine Greco, B., Lemmon, S., Pingchang Chow, E. and Kirchhausen, T.: The medium chains of the mammalian clathrin-associated proteins have a homolog in yeast. Eur. J. Biochem. 202 (1991) 569-574. Pearse, B.M.F.: Receptors compete for adaptors found in plasma membrane coated pits. EMBO J. 4 (1985) 2457-2460. Pearse, B.M.F. and Robinson, MS.: Purification and properties of 100-kd proteins from coated vesicles and their reconstitution with clathrin. EMBO J. 3 (1984) 1951-1957. Pearse, B.M.F. and Robinson, MS.: Clathrin, adaptors, and sorting. Annu. Rev. Cell Biol. 6 (1990) 151-171. Puszkin, S., Kohtz, J.D., Schook, W.J. and Kohtz, D.S.: Clathrin-coated vesicle subtypes in mammalian brain tissue: detection of polypeptide heterogeneity by immunopr~ipitation with monoclonal antibodies. J. Neurochem. 53 (1989) 51-63. Rupp, F., Payan, D.G., Magill-Sole, C., Cowan, D.M. and Scheller, R.H.: Structure and expression of a rat agrin. Neuron 6 (1991) 811--X23. Sambrook, J., Fritsch, E.F., and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Schmid, S.L.: The mechanism of receptor-mediated endocytosis: more questions than answers. BioEssays 14 (1992) 589-596. Serafini, T., Stenbeck, G., Brecht, A., Lottspeich, F., Orci, L., Rothman, J.E. and Wieland, F.T.: A coat subunit of Golgi-derived nonclathrin-coated vesicles with homology to the clathrin-coated vesicle coat protein @-adaptin. Nature 349 (1991) 2155220. Sosa, M.A., Schmidt, B., von Figura, K. and HilIe-Rehfeld, A.-H.: In vitro binding of plasma membrane-moated vesicle adaptors to the cytoplasm% domain of lysosomal acid phosphatase. J. Biol. Chem. 268(1993)12537-12543. Thurieau, C., Broius, J., Burne, C., Jolles, P., Keen, J.H., Mattaliano, R.J., Chow, E.P., Ramachandran, K.L. and Kirchhausen, T.: Molecular cloning and complete amino acid sequence of AP50, an assembly protein associated with clathrin-coated vesicles. DNA 7 (1988) 6633669. Virshup, D.M. and Bennett, V.: Clathrin-coated vesicle assembly polypeptides: physical properties and reconstitution studies with brain membranes. J. Cell Biol. 106 (1988) 39-50. Volknandt, W. and Zimmermann, H.: Identical properties of transmembrane synaptic veside protein M, l~,~ in Torpedo and &f, 86,000 in bovine brain. Neuroch~m. Int. 16 (1990) 5399547. Waters, M.G., Serafini, T. and Rothman, J.E.: “Coatomer”: a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles. Nature 349 (1991) 248-251. Zaremba, S. and Keen, J.H.: Assembly polypeptides from coated vesicles mediate reassembly of unique clathrin coats. J. Cell Biol. 97 (1983) 1339-1347.