A class of membrane proteins with a C-terminal anchor

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~i:i~i~i~! ! ANDERSON,R. G. W. (1989)/. Clin. Invest. 84, 1379-1386 8 KAMEN,B. A., WANG,M. T., STRECKFUSS,A. J., PERYEA,X. and ANDERSON,R. G. W. (1988)I. Biol. Chem. 263, 13602-13609 9 ROTHBERG,K. G., YING,Y-S.,KOLHOUSE, J. F., KAMEN,B. A. and ANDERSON,R. G. W. (1990) J. Cell Biol. 11 O, 637-649 10 ANDERSON,R.G. W., KAMEN,B. A., ROTHBERG,K. G. and LACEY,S.W. (1992)Science255, 410-411 11 YING,Y-S.,ANDERSON,R. G. W. and ROTHBERG,K. G. Cold Spring Harbor SFmp. Quant. Biol. (in press) 12 ROTHBERG,K. G. et ol. (1992) Ce1168,673-682 13 ROTHBERG,K. G., YING,Y-S.,KAMEN,B. A. and ANDERSON, R. G. W. (1990)]. Cell Bi0!. 111,293!--2938 14 CHANG,W-J.,ROTHBERG,K. G., KAMEN,B. A. and ANDERSON,R. G. W. (1992)l. Cell Biol. 118, 63-69 15 CINEK,T. and HOREISi,V. (1992)I. ImmunoL 149, 2262-2270 16 LOW,M. G. (1989) Biochim. Biophys. Acta 988, 427-454 |7 FRICK,G. P. and LOWENSTEIN,J. M. (1978)]. Biol. Chem. 253,

A class of membrane proteins with a Co.terminal anchor

Integral membrane proteins are generally targeted to translocation-competent membranes by virtue of signal sequences located close to the N.terminus of the polypeptide chain. Membrane anchoring is caused by the signal sequence or other hydrophobic segments located after it in the amino acid sequence. However, some integral membrane proteins do not follow these rules. The membeis of one class of nonconformist membrane proteins have no signal sequence, but instead possess a hydrophobic segment near the C-terminus that orients them with their N-termini in the cytoplasm. Members of this class are found in many organelles and are probably inserted into membranes by an unusual mechanism.

Most eukaryotic integral membrane proteins are inserted into translocation-competent membranes, such as those of the endoplasmic reticulum (ER), mitochondria or chloroplasts, during their biosynthesis, and they are anchored in the phospholipid bilayer by one or more hydrophobic segments in 72

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1240-1244 18 SASAKI,T., ABE,A. anti SAKAGAMI,T. (1983)1. Biol. Chem. 258, 6947-6951 19 CHE,M., NISHIDA,T., GATMAITAN,Z. and ARIAS,I. M. (1992) I. Biol.Chem.267, 9684-9688 20 HELTIANU,C., DOBRILA,L., ANTHOE,F. and SIMIONESCU,M. (1989) Microvasc. Res. 37, 188-203 21 MERIDA,I., PRAI"F,J. C. and GAULTON,G. N. (1990) Proc. Natl Acad. ScL USA87, 9421-9425 22 SALTIEL,A. R. and SORBARA-CAZAN,L. R. (1987) Biochem. Biophys. Res.Commun. 149, 1084-1092 23 PLOURDE,R., D'ALARCAO,M. and SALTIEL,A. R. (1992) I. Org. Chem. 57, 2606-2610 24 SUZUKI,S. and SUGI,H. (1989) Cell TissueRes.257, 237-246 25 FUJiMOTO,T., NAKADE,S., MIYAWAKI,A., MIKOSHIBA,K. and OGAWA,K. (1992)J. Cell Biol. 119, 1507-1514 26 DAVENPORT,R.W. and KATER,S. 8. (1992) Neuron9, 405-416

the polypeptide chain, called membrane anchors or stop-transfer sequences. They may then be transported to organelles that do not contain a translocation apparatus, such as the Golgi complex or plasma membrane. The processes of protein insertion into membranes and complete translocation across membranes seem to be mechanistically similar. For example, both ER membrane proteins and secretory proteins have similar hydrophobic signal sequences that are recognized by the signal recognition particle (SRP) during polypepttde elongation (see Ref. 1 for a review). The nascent chains must be at ]east 60 amino acid residues long before the N.terminal signal sequence emerging from the ribosome becomes accessible to the SRP2. During the translocation of a polypepqde across the ER membrane, which begins after docking of the complex of ribosome-bound nascent chain and SRP onto the membrane, other translocation components comm o n to both membrane and secretory proteins seem to be involved (see Ref. 1 for a review). However, it is possible that membrane proteins require additional components for their insertion, such as receptors for stop-transfer sequences3. Mitochondrial proteins transported across or inserted into the inner membrane also share translocation components (see Ref. 4 for a review). In addition, at least some proteins of the outer mitochondrial membrane appear to have signals at their N-term i n i similar to those that target other proteins to the matrix s. By contrast, a class of integral membrane proteins that have their membrane anchors at the Cterminus does not seem to follow these general rules. The archetype member of this class is the ERform of cytochrome bs, which has long been noted to be exceptional in its behaviour 6-8. It is cytoplasmically oriented and anchored in the membrane by a hydrophobic C-terminal sequence that has been called an 'insertion sequence' to distinguish it from a signal sequence 9. Although structurally TRENDS IN CELLBIOLOGYVOL. 3 MARCH 1993

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TABLE i - MEMBRANE PROTEINS WITH A C-TERMINAL ANCHOR a

similar to signal sequences, this insertion sequence does not compete with them for translocation even in a lO00-fold molar excess 10. Furthermore, SRP is not required for membrane integration of cytochrome bs (Ref. 11). Membrane

topology

We have assembled a list of proteins that appear to belong to this class of membrane proteins, based mainly on a systematic computer search of the Swissprot data base (Table 1). In this article, we summarize their characteristics and consider possible mechanisms for their insertion into membranes. All proteins listed in Table 1 have single hydrophobic membrane-anchor segments close to their C-termini. For many, the cytoplasmic location of the N-terminus has been demonstrated by its accessibility to proteases or to antibodies. This orientation (Fig. 1) makes it unlikely that N-terminal signal sequences have been overlooked. The insertion of these proteins into the membrane must occur post-translationally, since their single hydrophobic segment is too close to the C-terminus (within the last 50 residues) to have emerged from the ribosome before termination of translation. For such proteins of the ER, it also follows that SRP cannot be involved in their membrane targeting, since SRP requires the nascent chain to be bound to the ribosome 12. The proteins listed in Table 1 are firmly bound to the membrane: where tested, they cannot be extracted by high salt or urea and are often resistant to alkali 13A4. However, it is not clear from the literature whether they span the membrane with their C-termini on the extracytoplasmic side, or if they have a loop structure with the hydrophobic segment only 'dipping' into the phospholipid bilayer (Fig. 1); for example, the location of the C-terminal amino acids of cytochrome b s is under dispure 1s-17. For some proteins a loop structure seems unlikely since their membrane anchors are similar to classical transmembrane segments (i.e. about 20 hydrophobic residues flanked by charged ones). In any case, all members of the class would have at most only a few amino acids on the extracytoplasmic side of the membrane.

Cellular location Tail-anchored proteins have been found in several cellular compartments (see Table 1). The list includes a number of proteins in organelles of the secretory pathway, but it is not clear whether they are all inserted initially into the ER membrane. Interestingly, several tail-anchored proteins seem to be involved in vesicular transport [e.g. BOS1, BET1 (Ref. 18) and SLY2 (Ref. 19) of Saccharomyces cerevisiae, and the synaptobrevins2°]. The list also includes one protein located in the outer mitochondrial membrane and one in the nuclear membrane. Some tail-anchored proteins seem to have specific locations in the cell, while others, like the middle T antigen of polyoma virus, have been reported to reside simultaneously in several organelles 21. Although multiple cellular locations cannot

Protein

Source

Cellular location

Cytochrome bs

Mammals, birds

ER

Heme oxygenase

Mammals

ER

Microsomal aldehyde dehydrogenase

Rat

ER

Protein tyrosine phosphatase 1B

Human, rat

ER

T-cell protein tyrosine phosphatase

Human

ER (?)

UBC6b

S. cerevisiae

ER

Ramp2c

Dog, human

ER

Ramp4c

Rat

ER

SCYSY6

5. cerevisiae

ER

DPM1

5. cerevisiae

ER (?)

BOS1

S. cerevisiae

ER, ER-to-Golgi transport vesicles

BET1 (SLY12)

S. cerevisiae

ER, ER-to-Golgi transport vesicles (?)

SLY2

5. cerevisiae

ER-to-Golgi transport vesicles (?)

SED5

5. cerevisiae

ER-to.Golgi transport vesicles (?)

Phospholamban

Mammals, birds

Sarcoplasmic reticulum

Bcl-2

Human

ER, small amounts in plasma membraned

Middle T antigen

Polyoma virus

Plasma membrane, ER, other membranes (?)

NSP1

S. cerevisiae

Plasma membrane (?)

Synaptobrevins

Mammals, fish,

Small cytosolic vesicles

Syntaxin

Rat

Presynaptic plasma membrane

OMcytochromeb 5

Rat

Outer mitochondrial membrane

KARl

5. cerevisiae

Nuclear membrane

SCPEP12P

S. cerevisiae

Vacuolar membrane,

Epstein-Barr virus

?

Drosophila

Golqi (?) BHRF-1

aMost proteins in this class have been identified by a computer search of the Swissprot data base. The list contains polypeptides with a single hydrophobic segment of 16-20 amino acids that is located within the 50 C-terminal residues. For some proteins, like SCYSY6, SLY2 and NSP1, only the corresponding genes have been reported. A full list of references can be supplied by the authors on request. The recently discovered VIP21 (mammals, birds) may belong to the same class and is located in Go!gi-derived vesicles32. Monoamine oxidase B, located in the outer mitochondrial membrane, was also found in the computer search. It has a C-terminal targeting sequence33 but seems to be oriented with its N-terminus inside the mitochondrion 34. bT. Sommer and S. lentsch, pets. commun. cRibosome.associated membrane protein (D. G6rlich, E. Hartmann, S. Prehn and T. A. Rapoport, unpublished). dA location in the inner mitochondrial membrane has also been suggested.

73 TRENDS IN CELLBIOLOGYVOL. 3 MARCH 1993

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C

I

IJ

extracytoplasmic space i

membmne

cytoplasm

FIGURE 1 Possible membrane topologies of proteins with C-terminal anchor sequences. The C-terminal hydrophobic sequence may either span the phospholipid bilayer with few amino acids on the extracytoplasmic side (left) or, less likely, 'dip' into the membrane in a loop structure (right).

be excluded in some cases, such proteins are not present in all membranes, at least not in similar concentrations. The hydrophobic C-terminus is responsible for membrane anchoring, since its removal generally results in the appearance of the truncated protein in the cytoplasm13, 22-26. In intact cells, at least in some cases, the C-terminal sequence seems to be sufficient for targeting]3,2s, 27. Thus, when the tail of protein tyrosine phosphatase 1B or of cytochrome b s was attached to heterologous proteins, these fusion products were directed to the ER membrane13, 27. Also, the plasma membrane location of polyoma middle-T antigen can be disturbed by certain mutations in the hydrophobic domain2S. Transfection experiments have indicated that the last ten amino acids of cytochrome b s are important for its targeting to the ER although not all of them are hydrophobic 27. These data indicate that the C-terminal anchors are specifically recognized in the cell and direct the proteins to their destinations. On the other hand, there is evidence that, in vitro, some of the tail-anchored proteins, like cytochrome b s (Refs 15, 23), synaptobrevin and UBC6 (U. Kutay, unpublished), can insert into any membrane and even into liposomes. Thus, it seems that the proteins may interact only with the phospholipids of membranes, presumably through their hydrophobic tails.

The authorsare at the Max. DelbriJck.Center for Molecular Medicine, Robert-R6ssleStrasse10, 0-1115 Berlin-Buch,FRG. 74

Possible mechanisms of targeting How can one reconcile these conflicting results? By contrast to the situation in vitro, a newly synthesized protein in eukaryotic cells may encounter many different membranes and organelles. Although, in principle, it could be inserted into any membrane, several mechanisms can be envisaged to localize a protein of this type to a particular cellular membrane. One possibility is that receptors in the target membrane might increase the rate and/or extent of integration. The receptors might interact transiently with a tail-anchored pro-

tein, or they might associate permanently with it to form an oligomeric membrane protein complex. It is also conceivable that cytoplasmic factors bind to the newly synthesized protein to prevent its nonspecific insertion into membranes. This factor would have to be released when the correct target membrane was encountered. A further possibility is that the proteins indeed insert initially into many membranes but are then concentrated into a specific one by redistribution. Again, one would have to postulate specific binding partners in the target membranes. Further possibilities include special mechanisms for transport of the tailanchored proteins to their final destination, for example along elements of the cytoskeleton, or their synthesis at defined places in the cell by sequestration of their mRNAs. Finally, it seems plausible that mistargeted proteins are simply degraded by proteases. Other eukaryotic membrane proteins, with different topologies to that of the class of tailanchored proteins discussed here, also integrate into membranes by unusual mechanisms. One example is the a subunit of the SRP receptor, which has a membrane anchor at its N-terminus that does not function as a signal sequence28, 29. Rather, the protein is inserted into the ER membrane by an SRP-independent mechanism, perhaps involving an interaction with the [3 subunit of the SRP receptor 30. The N-terminally anchored NADHcytochrome bs reductase has been proposed to follow a similar route 31. A common denominator of these unusual mechanisms of membrane integration may be the fact that only few amino acids, if any, have to be transferred across the membrane. References 1 RAPOPORT,T. A. (1992) 5cience258, 931-936 2 WALTER,P. and BLOBEL,G. (1982) J. CeUBiol.91,557-561 3 LINGAPPA,V. R. (1991) Cell4, 527-530 4 PFANNER,N. and NEUPERT,W. (1990) Annu. Rev. Biochem. 59, 331-353 S HURT,E. C., MOLLER,U. and SCHATZ,G. (1985) EMBOJ. 4, 3509-3518 6 STRITUMATrER,P., ROGERS,M. ]. and SPATZ,L. (1972) J. BioL Chem. 247, 7188 7 RACHUBINSKI,R. A., VERMA,D. P. S. and BERGERON,]. ]. M. (1980) J. Cell BioL 84, 705-716 8 FLEMING,P. I. and STRITTMATFER,P. (1978)J. Biol. Chem. 253, 8198-8202 9 BLOBEL,G. (1980) Proc.NatlAcad. Sci.USA 77, 1496-1500 10 BENDZKO,P., PREHN,S., PFEIL,W. and RAPOPORT,T. A. (1982) Eur.1. Biochem. 123, 121-126 11 ANDERSON,D. ]., MOSTOV,K. E. and BLOBEL,G. (1983) Proc. Natl Acad. Sci. USA 80, 7249-7253 12 WIEDMANN,M., KURZCHALIA,T. V., BIELKA,H. and RAPOPORT,T. A. (1987) J. Cell Biol. 104, 201-208 13 FRANGIONI,J. V., BEAHM,P. H., SHIFRIN,V., lOST,C. A. and NEEL, B. G. (1992) Cell 68, 545-560 14 NEWMAN,A. P., GROESCH,M. E. and FERRO.NOVICK,S. (1992) EMBOJ. 11, 3609-3617 15 TAKAGAKI,Y., RADHAKRISHNAN,R., WIRTZ,K. W. A. and KHORANA,H. G. (1983) J. Biol. Chem. 258, 9136-9142 16 OZOLS,J. (1989) Biochim. Biophys. Acta 997, 121-130 TRENDS IN CELL BIOLOGYVOL. 3 MARCH 1993

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17 ARING,E., RZEPECKI,L. M. and STRITFMATFER,P. (1987) J. Biol. Chem. 262, 15563-15567 18 NEWMAN,A. P., SHIM.,J. and FERRO-NOVlCK,S. (1990) Mol. Cell. Biol. 10, 3405-3414 19 DASCHER,C., OSSIG,R., GALLWlTZ,D. and SCHMITT,H. D. (1991) MoL Cell. Biol. 11,872-885 20 SCHIAVO,G. et al. (1992) Nature 359, 832-835

21 DILWORTH,S. M., HANSSON,H. A., DARNFORS,C., BJURSELL,G., STREULI,C. H. and GRIFFIN,8. E. (1986) EMBO I. 5, 491-499 22 MASAKI,R., YAMAMOTO,A. and TASHIRO,Y. (1991) in Nato Conference on Protein Translocation in the Cell (Mizushima, S., Tubal, S. and Omura, T., eds), p. 73 23 DAILY,H. A. and STRITI'MATI'ER,P. (1978) J. Biol. Chem.253, 8203-8209 24 CHEN-LEW,Z. and CLEARY,M. L. (1990) I. Biol. Chem. 265, 4929-4933

A structural similarity between mammalian and yeast transcription factors for cell-cycle-regulated genes In the budding yeast Sacchoromyces cerevisiae, early cell cycle events are controlled by, and coordinated with, transcription by two distinct multicomponent transcription factors, called SBF and DSC1/MBF. These activate transcription of genes expressed during G1 and S phase by binding to two related sequence motifs, the SCB and MCB elements1 (Table 1). The DNA-binding specificity of SBF is provided by the N-terminal region of the SWI4 protein2, whereas the sequencespecific binding component in DSC1/MBF has yet to be characterized. However, both SBF and DSC1/MBF contain another protein, SWI6, that lacks intrinsic DNA-binding specificity but is necessary for proper transcriptional regulation 1. The cdcl 0 protein of the fission yeast Schizosocchoromyces pombe contains regions of similarity with SWI4 and SWI6 (Ref. 2) and is also found in a DSC1-like activity 3. The conservation of these functions between the distantly related budding and fission yeasts suggests

25 MARKLAND,W., CHENG,S. H., OOSTPA,B. A. and SMITH,A. E. (1986) J. Viral 59, 82-89 26 CARMICHAEL,G. G., SCHAFFHAUSEN,B. S., DORSKY,D. I., OLIVER,D. B. and BENJAMIN,T. L. (1982) Proc. Nail Acad. ScL USA 79, 3579-3585 27 MITOMA, J. and ITO, A. (1992) EMBOJ. 11, 4197-4203 28 HORTSCH,M. and MEYER,D. I. (1988) Biochem. Biophys. Res. Commun. 150, 111-117 29 ANDREWS,D. W., LAUFFER,L., WALTER,P. and LINGAPPA,V. R. (1989)I. Cell Biol. 108, 797-810 30 TAJlMA,S., LAUFFER,L., PATH,V. L. and WALTER,P. (1986) I. Cell Biol. 103, 1167-1178 31 BORGESE,N. and LONGHI,R. (1990) Biochem. J. 266, 341-347 32 KURZCHALIA,T. V. et al. (1992)]. Cell Biol. 118, 1003-1014 33 MITOMA,J.and ITO, A. (1992)1. Biochem. 111, 20-24 34 ZHAUNG,Z. and MCCAULEY,R. (1989)I. Biol. Chem. 264, 14594-14596

that other organisms may use similar mechanisms to regulate transcription during ceil cycle progression. In higher eukaryotic cells, the DRTF11E2Ftranscription factor has been proposed to perform an analogous role to the yeast SBFand DSC1 systems4 because it is responsible for the periodic transcription of genes that, in some cases, are necessary for cell cycle progressions and its transcriptional activity is regulated by proteins, such as the retinoblastoma gene product (pRB) and the pRB-related protein p107 (Ref. 6), that lack intrinsic sequence-specific DNA-binding activity. Furthermore, the DNA sequences of SCBs, MCBs and E2F-binding sites have a similarity (see Table 1) that suggests structural conservation of the protein domains that recognize them. We tested this idea by comparing the DNA.binding domains of SWI4 and cdcl0 (Ref. 2) with two recently characterized proteins isolated from mammalian cells, F.2F-1 (Refs 7, 8) and DP-1 (Ref. 9), that specifically recognize the E2F-binding site.

....

A comparison of the sequences of E2F-1 and DP-1 revealed a region of similarity within their DNA-binding domains, while the rest of these proteins were very different 9 (Fig. 1). The conserved amino acid residues in this region suggested that it may be similar to a region within the SWI4 and cdcl0 DNA-binding domains, and a motif derived from this region in all four sequences consisted of a conserved pair of positively charged residues, followed by a region of alternating hydrophobic and hydrophilic residues, followed again by a more basic region containing a few hydrophobic positions (Fig. 1). With the inclusion of gaps, a further region of weaker similarity could be found beyond the main motif (indicated in Fig. 1). Overall, the conservation of sequence is weak and includes only two completely conserved positions, Arg3 and Asnl 0. A motif derived from the first 18 positions was taken as the core pattern lo with which to search the protein sequence data banks for matching patterns. The principle of pattern matching relies on defining a pattern of amino

TABLE 1 - COMPARISON OF MCBS, SCBS AND E2F-BINDING SITES DNA motif

Organism

DNA sequence

Binding activity

Sequence-specific protein

MCB MCB SCB E2F-binding site

S. pombe S. cerevisiae S. cerevisiae

Mammals

TNACGCGT TNACGCGT

TTTTCGTG TTTCGCGC

DSC1-like DSC1/MBF a SBF r nTC'I" . . . . . /~2F

cdcl 0 p120 SWI4 DP-1 and E2F-1

aThe DSC1/MBF p120 DNA-binding polypeptide (Ref. 12; L. Johnston, pers. commun.) has not been characterized.

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