Proteins involved in vesicular transport and membrane fusion

Proteins involved in vesicular transport membrane fusion M. Gerard

Waters, Princeton

and

Irene C. Griff and James E. Rothman University,

Princeton,

New Jersey, USA

In the past year, new information about proteins involved in vesicular transport has been plentiful. Particularly noteworthy are the complementary findings that Secl7p is required for vesicle consumption in endoplasmic reticulum-to-Go@ transport in yeast and that an analogous activity in mammalian cells, termed SNAP, is required for transport from the cis to the medial cisternae of the Colgi apparatus. Current

Opinion

in Cell Biology

Introduction

formed detailed electron microscopic analysis of a subset of these ER-to-Go@ transport mutants and found that they can be divided into groups that either do, or do not, accumulate approximately 50 nm vesicles that are believed to be intermediates in ER-to-Go@ transport. Specifically, the mutants secl2, secl.3, secZ6, and sec21 (known as class I mutants) do not accumulate vesicles at the non-permissive temperature, whereas the mutants secZ8, secZ7, and sec22 (known as class II mutants) accumulate transport vesicles. In addition, inspection of double-mutant morphology has indicated that class I mutants prevent vesicle accumulation by class II mutants. This epistasis indicates that class I gene products are required for vesicle formation and that class II proteins are involved in vesicle consumption. The interpretation that SeclSp is required for vesicle consumption is in agreement with the linding [ 111 that a protein, N-ethylmaleirnide-sensitive fusion protein (NSF) is the mammalian equivalent of Secl8p [ 121. NSF is involved in consumption (fusion) of Golgi transport vesicles [ 131 and fusion of endocytic vesicles, and is required for ER-to-Go@ transport in a mammalian ER-to-Go@ transport system [ 141.

The mechanism of protein and lipid transit through the secretory and endocytic pathways is of fundamental importance in cell biology. In general, proteins-are transported in vesicles that bud from one membrane compartment and then specifically target to, and fuse with, another [ 11. Several approaches have been used to study this process. Initially, morphological studies [2] defined the problem, and they have been used since to study the compartments involved in transport. Another approach, which has proved very successful, has been to generate and study vesicular tralhc mutants in the yeast Sac&zromyces cerez)biae [3,4]. A third approach has been to reconstitute specific transport steps in vitro [5] and to dissect these systems biochemically. In the last year, each approach has yielded interesting findings, and in one case there has been a contluence of these approaches leading to insight into the mechanism of transport-vesicle consumption, that is, fusion with the target membrane [6-,7-l.

Genetic analysis of vesicle consumption in yeast

production

In addition to the epistatic relationship between the class I and class II mutations, there are genetic interactions within each group [7**]. This was found by noting that although strains that bore single class I or class II mutations were able to grow at the permissive temperature, certain double mutants were inviable under the same conditions. This phenomenon, termed synthetic lethality [15], was only found when both mutations were within the same class. Specifically, secl2, secl3, secl6, and sec23, which are believed to be involved in vesicle formation, exhibited synthetic lethality in five out of the possible six double mutants. Of the vesicle-consumption mutants, secl7 and secl8 double mutants were inviable. Synthetic lethality

and

Genetic analysis of secretion in yeast has shown that some secretion mutants accumulate a membranous organelle (for example, endoplasmic reticulum (ER) or Golgi) at the non-permissive temperature, while others accumulate vesicles, and yet others both an organelle and vesicles [3,4,8]. A number of these mutants block transport of protein from the yeast ER to the Golgi complex [9.,10*]. Recently, Raiser and Schekman [7*-l per-

COP-coat

protein; ER+ndoplasmic PI-phosphatidylinositol;

Abbreviations reticulum; NSF-N-ethylmaleimide-sensitive SNAP--soluble NSF attachment @

Current

Biology

1991, 3:61S-620

Ltd

ISSN

protein; 0955*74

fusion protein; TCN-trans-Colgi

PCyhosphatidylcholine; network. 615

616

Membranes

and suppressor analysis also suggest protein interactions between the Sec22, Betl, and Bosl proteins [ 16*].

Fractionation of the cytosolic required for Golgi transport

components

Vesicular transport through the Golgi apparatus has been ar&ysed biochemically and morphologically and shown to require membrane and cytosolic proteins, as well as ATP and palmitoyl-coenzyme A [ 17*]. Subfractionation of the cytosolic components required for vesicular transport from the cb to the medial cistemae of the Golgi complex in v&-o, has shown that both high ( > 100 kD) and low ( < 100 kD) molecular weight components are required for the reaction [ 18.1. The low molecular weight activity has been purified to homogeneity and found to be composed of a family of proteins of 35,36, and 39 kD. These proteins, each of which is active alone, can bind to Golgi, and in doing so, bind NSF to the membrane [6**,19]. The 35, 36, and 39kD soluble NSF attachment proteins (SNAPS) are termed a-SNAP, @%AP, and y-SNAP, respectively. In agreement with the Snding that NSF acts late in the Golgi transport reaction [ 131, after targeting of the transport vesicle to the acceptor membrane and removal of the vesicle’s coat [20], it has been found that the activity of SNAP is also required late [6-l. (Note that a requirement in vesicle consumption does not preclude a function in vesicle formation.) The connection to the genetic and morphologic studies of Kaiser and Schekman described earlier was made possible by the finding of Clary ef al. [6**] that yeast cytosol could provide SNAP activity in the mammalian system. This allowed the screening of secretion-mutant cytosols in the assay with the result that secl7 mutant cytosol was found to be deficient in SNAP activity; a-SNAP, but not j!ML4P or y-SNAP, was able to complement the defect in xc27 cytosol. The ability of SNAP (i.e. mammalian Secl7p) to bind NSF (i.e. mammakan SeclBp) to the Golgi membrane supports the interpretation that the synthetic lethality exbibited by secl7/sec18 double mutant is a result of the ‘amplification of the defects in the individual mutants when their protein products physically interact [7-l.

Proteins

involved

in vesicle

formation

Another method for analysis of proteins involved in vesicle formation is through production of vesicles using in vitro systems [ 21,22,23*,24*]. These systems bud vesicles from a suitable donor membrane in the presence of ATP and cytosol. Permeablized yeast cells have been used to

this end for production of ER-to-G&j vesicles [23*] and have yielded a crude vesicle fraction that is active, i.e. that can be separated from the donor ER and then targeted to, and fused with, acceptor Golgi. Purification of these vesicles should yield insight into their components. In addition, as the vesicles are active, the requirements for vesicle production (and subsequently for vesicle consumption) can be assessed by fractionation of the cytosolic components required for production and/or by analysis of whether secretory mutant cytosol can drive vesicle production and/or consumption. Two types of vesicles that bud from the tru~Golgi network (TGN) in a polarized cell and are subsequently targeted to either the apical or basolateral plasma membrane domains have been purified in the last year [25*]. Two-dimensional gel electrophoresis of the vesicle proteins in conjunction with Triton X-114 phase partitioning indicated that the two types of vesicles contain common, as well as distinct, components. The common components were postulated to be involved in the general budding and fusion mechanism, whereas proteins specific to one vesicle or the other were suggested to be involved in protein sorting or targeting. Golgi transport reactions accumulate a population of coated vesicles when performed in the presence of the non-hydrolyzable GTP analog, GTP-YS [ 261. These vesicles, believed to be involved in Golgi transport, have been purified after in vitro production from mammalian Golgi [ 221. Although the vesicles bear a cytoplasmic coat, it is not composed of clathrin [ 22,27,28] as is the coat of vesicles that bud from the plasma membrane during endocytosis or from the TGN for delivery of proteins to the lysosome [ 291. Recently, improvements in the purification scheme have allowed Golgi transport vesicles to be obtained with a very simple protein pattern, consisting of pomptides of 160, 110,98, and 61 kD, and a number of lower molecular weight components [30-l. The largest coat proteins are termed a-COP, P-COP, y-COP, and &COP, respectively. The proteins are present on the cytoplasmic face of the transport vesicles and are therefore believed to represent the subunits of the cytoplasmic coat evident on Golgi transport vesicles. One of the coat proteins, P-COP, was previously identified as a Golgi peripheral membrane protein [31] using immunofluorescence and cell fractionation. lmmunoelectron microscopy localizes P-COP to the Golgi complex and associated transport vesicles in vivo and in vitro [30**,32**]. The P-COP cDNA has been cloned and the sequence determined [32-l and it was found that P-COP is homologous to a protein of similar size in clathrincoated vesicles, termed p-adaptin, which is part of a complex that links the clathrin lattice to the underlying vesicle membrane. The sequence homology suggests that pCOP and p-adaptin may have evolved from a common ancestral protein. This raises the exciting possibility that the two known types of coated vesicles, previously believed to be unrelated, may exhibit underlying similarities in structure and assembly.

Proteins

involved

in vesicular

It has been found that P-COP, which has a molecular weight of 110 kD, is present in cytosol in a complex of approximately 6OOkD molecular weight [32**,33**]. This complex, which has been termed ‘coatomer’, has been purified and found to consist of a-COP, P-COP, y-COP, S-COP, and proteins of 36, 35, and 20 kD [33**]. The coatomer represents approximately 0.2% cytosolic protein, an amount similar to that of clathrin in several cell types [34]. Although it has been suggested that the coatomer is required for vesicle formation, this remains to be demonstrated directly. Apart from the requirement for cytosol and ATP in in vitro vesicle-formation reactions, it has been demonstrated that GTP is required for formation of secretory vesicles and immature secretory granules from the TGN

L35.1.

Proteins

transport

and membrane

fusion

membrane [3]. Several of these have been characterized, namely the GTP-binding protein Sec4 [43], and the Secl5 protein [441. Recently, Novick and coworkers (45’1 have described some properties of the Sec2 protein. This protein contains an amino-terminal coiled-coil domain that is essential for vesicular transport in vivq it seems to bind to other polypeptides, possibly through the coiledcoil domain, as the soluble Sec2 protein shows a native molecular mass much greater than that predicted by its polypeptide sequence. sec2 exhibits synthetic lethality with the same group of late-acting set mutants, as do sec4 and secZ5. These Set proteins may be components of the native Sec2 protein complex.

Proteins involved in transport to the vacuole in yeast

involved

In vesicle

consumption

A number of proteins discussed earlier - NSF (Secl8p) and SNAP (Secl7p) - and the product of the SEC22 gene are involved in vesicle consumption. In addition, several reports have indicated that GTPbinding proteins may be involved in the mechanism of vesicle fusion in both the secretory [36*] and endocytic pathways [37*-391 (see review by Goud and McCaffrey, this issue, pp 626-633).

Other proteins transport

involved

in vesicular

The yeast mutant, secl4, has been shown to interfere with protein transit through the Golgi apparatus [3,40]. Recendy, SEC24 has been found to be identical with the gene, PITY, which encodes a phosphatidylinositol (PI)/phosphatidyicholine (PC) transfer protein [41**] that is capable of transfering either PI or PC between lipid bilayers (for review, see Downham, this issue, pp OOO-OOO).The exact role of this protein in transport through the Golgi apparatus remains to be determined. However, it has been suggested that it may be involved in either specilic retrieval of lipid from the Golgi apparatus or delivery to the ER [41*=,42*].

Proteins involved in transport from to the plasma membrane in yeast

Waters, Criff, Rothman

from

the Golgi

Several genetic selections have identified mutants which defme at least 47 complementation groups that are implicated in vacuolar protein sorting ( qs mutants) [ 46.1. These mutants have been characterized according to vacuolar morphology at the non-permissive temperature and many show defects in sorting of soluble vacuolar proteins. These vps mutations, however, also affect cell growth, vacuolar assembly, organellar pH, sporulation, and osmoregulation [46*]. It is not clear if the vacuolar sorting defect is the primary problem or if it is a consequence of these other phenotypes. Sequences of five of the vps genes have been published, with several showing interesting homologies [47*-5l*]. Characterization of the function of these proteins in vacuolar targeting will be facilitated by a recently described in vitro system that reconstitutes this process [52*]. The transport system, which is monitored by the processing of the Golgi form of carboxypeptidase Y to the vacuolar form, requires ATP, a cytosolic fraction, and active processing enzymes in the yeast vacuoles. The Vpsl5,33 and 34 membrane preparations are inactive, while Vpsl5 and 33 cytosolic fractions are active. This is consistent with the membrane association of Vpsl5p and Vps33p.

Conclusion During the past year there have been significant advances in the field of vesicular transport III the future, the combined application of genetics and in vitro biochemical and morphological analyses is certain to provide a more detailed understanding of this fundamental cellular process.

the Golgi

A number of SECgene products are required for movement of proteins from the Golgi complex to the plasma

Acknowledgements MG Waters is a Merck Sharp & Dohme Fellow of the Life Sciences Research Foundation.

617

618

Membranes

References

and recommended

readirig

14.

Papers of special imetes published within the annual period of review, hWbfXllhighUghtedaS: . of interest .. of outstanding interest 1.

P.UDE G: IntraceUuIar Aspects of the Process of Protein secretion. science 1975, 189347-358.

2.

CARO IG, PARADEGE: Protein charge graphic

3.

in the Pancreatic study. J cell Bid

NOMCK P, FIEID C. SCHE~ mentation the Yeast

4.

Synthesis, Storage, and DisExoaine Cd. An Autoradio1964, 20:473-495.

R: Identification of 23 CompleGroups Required for Post-Translational Events in Secretory Pathway. Ceu 1980, 21:205-215.

NEWMAN AP, FERRO-NOVICKS: Characterization tants in the EarIy Part of the Yeast Secretory

lated by a 13HlMannose Suicide Selection.

of New Pathway J Cdl Biol

MuIso-

1987,

105:1587-1594. 5.

Transport Faseb J

15. 16. .

Along

the

Secretory

Systems to Study and Endocytic

1989, 3:2488-2495.

CLUW DO, GIUPFIC. ROTHMANJE: SNAPs, a Family of NSF Attachment Proteins InvoIved in Intracellular Membrane Fusion in AnimaIs and Yeast. Cell 1990, 61:709-721. Three recently isolated proteins, termed a-, p-, and y-SNAP, were shown to function in Golgi vesicular transport at the fusion stage. Wdd-type yeast cytosol contained SNAP activity, whereas secl7 mutant cytosol did not - a condition that could be remedied by addition of a-, but not fi- or y-SNAP. 6. ..

KN.SERCA, SCHEKMANR Distinct Sets Of SEC Genes C&Vem Transport VesicIe Formation and Fusion Early in the Secretory Pathway. CeU 1990, 61:723-733. Morphological anal@ has identihed an accumulation of approximately 50 nm vesides in a subset of secretion mutanrs blocked in ER-to-Go@ nansport. This suggested that one set of gene products is involved in vesicle formation, tie the other is required for vesicle consumption. Double mutants indicated that vesicle formation is epistatic to vesicle consumption. Genetic interactions within each group suggested that the gene products may interact to perform their function,

17. .

R: Compartmentalized Exported Glycoproteins

Asin

Cytoskeleton

in Yeast?

Harvq,

NEWMANAP, SHIMJ, FERRO-NOVICK S: BET1, BOSI,

ROTHMANJE, ORCI L Movement Golgi Stack: A Molecular Diction FASEB J 1990, 4:1460-1468.

of

Proteins Through the of VesicuIar Transport.

CLUY DO, ROTIWAN JE: Purification of Three Related Peripheral Membrane Proteins Needed for Vesicular Transport. J Biol C5em 1990, 265:1010’+10117. cytosolic components required for tmnspon in vitro were shown

The to be separable into high and low molecular weight fractions. Purification of the low molecular weight activity yielded three proteins, each Sufficient to ECOnStiNte intra-Go@ transport in r&a 19.

WEIDMANPJ, MEWGON P, BLOCK MR ROTHMANJE: Siding of an N-EthyImaIeimide-Sensitive Fusion Protein to Go&i Membranes Requires Both tegral Membrane Receptor.

a Soluble Protein(s) J Cell Bioll989,

and

an In-

108:158+15%.

20.

ORCl I, MALHOTRAV, AhtHERDTM, SER4FlNlT, Ron-lhcw JE: Dissection of a Single Round of Vesicular Transport: Sequential Intermediates for IntercisternaI Movement in the GoIgi Stack. Cell 1989, 56:357-368.

21.

BENNETTMK, WANDLNGERw

S~ONS K: Release of Putative

Exocytic Transport Vesicles EMBO J 1988, 7:4075-4085.

From

..

FSION B, NOVICK P, SCHEW

the

82:157-167.

A concise review of morphological and biochemical analyses of transport of proteins through the Go@ apparatus.

7.

8.

D: Why Study

ing that these encode interacting genes products.

.

Vesicular Pathways.

B~TSTEIN Lea 1988,

ReticuIum Fusion Ro-

and SEC22 are Members of a Group of Interacting Yeast Genes Required for Transport From the EndopIasmic Reticulum to the GoIgi Complex. Md Cell Bid 1990, 10:340+3414. A new gene, BOSI, was isolated as a suppressor of the ER-toGo@ mutant, bell. Overproduction of either BET1 or BOSI suppresses the ERtoGolgi se&2 block. betl-sec22 double mutants are inviable suggest-

18.

GODA Y, PFF.FFER SR Cell-free

BECKERSCJ, BLXK MR, GUCK BS, ROTHMANJE, B.UCH WJZ: Vesicular Transport Between the EndopIasmic and the GoIgi Stack Requires the NEM-Sensitive tein. Nature 1989, 339:397-398.

22.

Perforated

MDCK

Cells.

M.UHOTR.4V, SEM T, ORCI L, SHEPHERDJC, ROTHMANJE: Purification of a Novel CIass of Coated Vesicles Mediating Biosynthetic Protein Transport Through the Golgi Stack. Cdl 1989, 58:329-336.

A short review of genetic and biochemical characterization of ER-toGo@ transport

GROESCHME, RUOHOLAH, BACONR, ROSSIG, FERRO-NOVICK S: Isolation of a Functional Vesicular Intermediate that Mediates ER to Go@ Transport in Yeast. J cell Biol 1990, 111:4553. A functional intermediate for ER-to-Go@ transport was produced in vitro using permeabilized yeast cells and then partiaUy purified. This in vitro system, because it separated vesicle formation from targeting and fusion, allowed the examination of the components required for both vesicle production and consumption.

10. .

.

11.

This paper describes an in vitro srjtem which is capable of forming both immature secretory granules and constitutive secretory vesicles from the TGN. Proteins destined for different vesicles were shown to be sorted correctly in this system.

sembly Yeast. 9. .

of Oligosaccharides cell 1981, 25:451-460.

on

I+ SCHEKMAN R Molecular Machinery Required for Protein Transport from the Endoplasmic Reticulum to the GoIgi Complex. BioEssay 1990, 12~253-258. HICKE

BMCH WE: Molecdar Dissection of Early Stages of the Eukaryotic Secretory Pathway. Curr Opin CkU Biol 1990, 2:634&Q. A brief review of biochemical and genetic studies of ER-to-Go@ trans. port. Wtt~o~ DW, WILCOX CA, FLYNN GC, CHEN E, KUANG WJ, HENZELWI, BLOCK MR, UURICH A, RCYIHMANJE: A Fusion Rotein Required MammaUan CeUs

for Vesicle-Mediated and Yeast. Natire

Transport in Both 1989, 339:35+359.

12.

EAKU KA, BERNSTEIN M, Ehta SD: Characterization of a Component of the Yeast Secretion Machinery: Identihcation of the SEC18 Gene Product. Md C&U Bioll988, 84098-4109.

13.

MAIHOIRA V, ORCI I, GUCK BS, BUICK MR, Ro~~lvrl JE: of an N-ethyImaIeimi&-sensitive Transport Component Promoting Fusion of Transport Vesicles with Cistemae the GoI@ Stack. cell 1988, 54221-227.

Role in of

23.

.

24.

25.

TOOZE .S& HUIINER WB: Cell-Free Rotein Regulated 60837-847.

WANDINGER

and

NA,

Constitutive

BENNETT

Secretory

hfK, ANKINY

Sorting to the Cell 1990,

Pathways.

C, SIMONS K: Distinct

Transport Vesicles Mediate the Delivery of Plasma Membrane Roteins to the Apical and BasoIateraI Domains of MDCK Cells. J Cell Bid 1990, 111:987-1000. This work characterizes two populations of transport vesicles formed from the TGN of polarized cells in oitm one type targeted to the apical plasma membrane domain, the other to the basolateral domain. Proteins common to both basolaterally and apicalIy targeted vesicles were proposed to be invoIved in general budding or fusion processes, whereas proteins unique to either type of vesicle were suggested to be tequired for targeting or protein sorting. .

Proteins 26.

involved

in vesicular

MRLANGON P, GUCK BS, MAIH~ V, WEIDMAN PJ, SEL%FINI T, GLEASON Ml, ORCI L, ROTHMANJE: Involvement of GTPBinding ‘G’ Proteins in Transport Through tbe Golgi Stack. cell 1987, 51:10531062.

27.

ORCI I, GUCK BS, ROTHMANJE: A New Type of Coated Vesicular Carrier that Appears not to Contain Clathrin: its Possible Role in Protein Transport Within the Golgi Stack. Cell 1986, 46:171-l&1.

28.

Gms G, PFEIFFER S, SIMONS K, MATU~’ K: Exit of Newly Synthesized Membrane Proteins From the Trans Cistema of the Golgl Complex to the Plasma Membrane. / CeN Biol 1985, 101:%+964.

29.

PEU?SEBMF, ROB~NSN MS: Clathrin, Adaptors, and Sorting. Annu Rev Cell Bid 1990, 6:151-171.

30. ..

SERAF~NIT, R~THMAN

STENBECK G, BRECHT A, LO-I-IXPEICH F, ORCI I JE, WELAND m: A Coat Subunit of Golgi-De-

rived Non-Clathrin-Coated Vesicles with Homology to the Clathrin-Coated Vesicle Coat Protein Beta-Adaptin. Nature 1991. 349215-220. Golgi-derived coated vesicles were generated in vitrw and highly purified. They were found to contain four high molecular weight coat pomptides, termed a-COP (160 kD), j3-COP (110 kD), y-COP (IlOkD), and &COP (61 kD), as well as sevetal smaller proteins. /3COP was shown to be identical to a protein with homology to a similarly sized protein of clathrin-coated vesicle, B-adaptin. 31.

AUAN VJ, KREISTE: A Microtubule-Binding Protein Associated with Membranes of the Golgi Apparatus. Ceil Biol 1986, 103222+2239.

32. ..

DUDENR, G-s G, FRANKR, ARGOSP, KREtsTE: B-COP, a 110 kD Protein Associated with Non-Clathrin-Coated Vesicles and the Golgi Complex, Shows Homology To BAdaptin. CeN 1991, 64:64%55. cDNA for a IlO-kD Go@-associated protein, B-COP, was cloned and sequenced and found fo encode a protein homologous to B-adapt@ a component of clathrin-coated vesicles. lmmunoelectron microscopy demonstrated that p.COP is localized to non-clathrin-coated vesicles and the Golgi apparatus. B-COP was shown to be present in cytosol in an approximatety 600.kD complex. WATERSMG. SERAFINI T, ROTHMAN JE: ‘Coatomer’: a CytosoIic Protein Complex Containing Subunits of Non-ClathrinCoated Golgi Transport Vesicles. Nature 1991, 349:24&251. An approximately 600 kD cytosolic complex containing a-COP, B-COP, and what is believed to y-COP and &COP, as well as 36, 35, and 20kD polypeptides was identified and purilied. This complex was termed the Go@ coat protomer or ‘coatomei and represents approximately 0.2% of soluble cytosolic protein. It was suggested that the coatomer serves as the assembly unit for the coat of Golgi-derived coated vesicles. 33. ..

34.

GOUD B, HUET C, LOWARD D: Assembled and Unassembled Fools of Clathrin: A Quantitative Study Using an Enzyme Immunoassay. J Cdl Biol 1985, 100:521-527.

TOOZE SA Requirement for GTF Hydrolysis in the Formation of Secretory Vesicles. Nutwe 1990. 347:207-208. ;t was shown the that non-hydrolysable GTP analog, GTP-yS, inhibits the formation of immature secretory granules and constitutive secretory vesicles from the TGN in an in t&-o system. It was suggested that GTPbinding proteins may be required for vesicle budding in addition to their previously proposed functions in vesicle targeting and fusion. 35.

PLUTNERH, SCHWANLNGER R, PONDS, BALCH WE Synthetic Peptides of the Rab EfTector Domain Inhibit Vesicular Transport Through the Secretory Pathway. EhlBO J 1990, 92375-2383. Synthetic peptide analogs of the putative etfector domain of Rab proteins, GTP-binding proteins of low molecular weight implicated in exocytic vesicular transport, were used to inhibit both ER-toGoI@ and inua-Go@ vesicular transport in virm Inhibition is temporally late, near fusion, and the site of peptide action is postulated to be a putative ‘Rab e.&ctor protein’ that may regulate vesicle fusion. 36. .

transport 37.

and membrane

fusion

Waters,

Griff,

G~RVEL J-P, CHAVRIER P, ZEIUAL M, GRUENBERG J: Rab5

Rothman Cootrob

Early Endosome Fusion In Vhtx cell 1991, 61:915-925. ke GTP-biding protein Rab5, which is kx&zed to the early endosome and plasma membrane, was shown to be invom in early endosome-eady endosome fusion in Tim The fusion reaction was stimulated by wild-type Rab5, but not by mutant forms of Rab5. Additionally, the Rab5 antibody inhibited the fusion reaction, but the inhibition could be overcome by addition of excess wild-type Rab5. WESSUNG RM, B~AEU WA Characterization of the Me&a&m 38. . of Endocytic Vesicle Fusion In Vjtrn J Bid &?m 1990, 265:16751-16759. A cell-free system was used to show that separate GTP-biding and ATP-binding proteins are required prior to involvement of an Nethylmaleimide-sensitive factor (not NSF) for endoqic wicle fusion. MAYORGAIs. DIAZ R, STAHL PD: Regulatory Role for GTP39. Binding Proteins in Endocytosis. Science 1989, 244:147> 1477. 40. NOV~CK P, FERROS, !XHEKMAN R: Order of Events in the Yeast Secretory Pathway. Cell 1981, 25461469. 41. ..

BAhXunsVA,ArrKENJR,CLEVESAE,Do\VHANW:AnEssencial Role for a Pbospholipid Transfer Protein in Yeast Golgi Function. Nature 1990, 347:561-562. Nucleotide and protein sequencing in conjunction with phenotypic and in vitro biochemical ana&s indicated that the genes SEC14 and PIT1 are allelk. SEClB/PITI encodes a phosphatidylinositol/phosphatidyicholine transfer protein that is required for secretory protein exit from the Golgi complex. The possible role of this 35kD protein in protein transit from the Golgi is discussed. 42. ROTHMANJE: Pbospholipid Transfer Market Nature 1990, . 347:519-520. A comment on the work of Bankaitis et al on the Secl4/Pitl phosphatidyiinositol/phosphatidylcholine transfer protein advocating the view that the function of this protein is to return lipid from the Golgi to the ER SNMNEN A, NOVICK PJ: A Ras-Like Protein is Required for a 43. Post-Go&i Event in Yeast Secretion. Cell 1987, 49527-538. SAMNEN 4 NOV~CKPJ: The Secl5 Rotein Responds to the 44. Function of the GTP Binding Rotein, Sec4, to Control Vesicular TralEc in Yeast. J Cell Biol 1989, 109:10231036. NAIRJ, MOUER H, PETERSONM, NOMCK P: Sec2 Rotein Con45. . tabs a Coiled-coil Domain Essential for Vesicular Transport and a Dispensable Carboxy-terminal Domain. J Cd Biol 1990, 110:1897-1909. Sec2 protein is shown to contain a coiled-coil domain which may mediate protein interactions with other SEC gene products. Genetic anaiysis suggests Sec2p may interact with Sec3p, Sec4p, SecSp, Sec8p, SeclOp, or SeclSp. 46. k4ONSKY DJ. HERMAN PK, EMR SD: Tbe Fut@ Vacuole. . Composition, Function, and Biogenesis. Microbid Rev 1990, 54:266-292. A comprehensive review of the assembly, targeting, and sorting of proteins to the fungal vacuole. The review also describes vacuolar ATPase and vacuolar acidilication. 47. ROTHMANJH, RAYMONDCK, GI~BER’TT, O’HARA PJ. STEVENSTH: . A Putative GTP Binding Protein Homologous to InterlizronInducible Mx Proteins Performs an Essential Function in Yeast Protein Sorting. cell 1990, 61:10631074. vpt.26~ (Vpslp) was shown to contain a GTP-binding consensus element, implying that GTP binding (and hydrolysii) is important for proper ~cuolar ttatTicking. The protein was also shown to be associated with the Golgi or post-Go@ organelles. 48. .

RAYMONDCK, O’HARA PJ, ETCHINGERG, Rm JH, S’IFVENS TM Molecular Analysis of the Yeast ITS3 Gene and the Role of its Product in Vacuoku Protein Sorting and Vacuolar mation During the Cell Cycle. J cell Bid 1990, 111:877-892. Characterization of the VP$? gene and its gene product show the Vps3p protein to be a 117 kD hydrophilic @peptide. Genetic and morphological analysis of vps3 mutants is also desctibed.

619

620

Membranes mu LM. Vro~ TA, HERMAN PK, Ehot SD: Characterization of Yeast Vps33p, a Protein Required for Vacuolar Protein Sotting and Vacuole Biogenesis. Mol Cell Biol 1990, 104638-4649. Vps33 protein was shown to be a 75kD cytoplasmic protein with sequence similarity to ATPases with two domains, each of which is pmdieted to bind and hydrolyse ATP. 49. .

50. .

HERMAN PK. EMR SD: Characterization of vPS34, a Gene Required for Vacuolar Protein Sorting and Vacuole Seg regation in Saccharomyces cerevfsiae. Mol Cell Biol 1990, 10:6742-6754. The predicted protein sequence of Vps34 indicated that it is a 95kD cytoplasmic polypeptide. It was found to be associated with the membrane fraction from lysed yeast cells via protein-protein interactions.

51. .

HERMAN PK. STACK JH, DEMODENA Protein Kinase Homolog Essential the Yeast Lysosome-Like Vacuole.

JA EMR SD: A Novel for Protein Sorting to Cell 1991. G4:425-437.

Vpsl5 was shown to be homologous to the Ser/Thr family of protein kinases. An active Vpsl5p kinase domain is shown to be essential for proper vacuolar protein localization and the phosphorylation of Vpsl5p itself Vpsl 5p contains an amino-terminal myristic acid that may mediate its stable association with a late Golgi compartment and/or vesicles. VIDA TA, GRAH~~( TR, Ehirr SD: In V&o Reconstitution of Intercompartmental Protein Transport to the Yeast Vacuole. J Cell Biol 1990, 2871-2t3B4. Describes an in t&-o assay that monitors transport from the G&i apparatus to the yeast vacuole. Several fpf mutants were examined for their role in this process.

52. .

MG Waters, IC GriR and JE Rothman, Program in Cellular Biochemistry and Biophysics, Rockefeller Research Laboratories, Sloan-Kettering Institute, 1275 York Avenue, New York, New York 10021, USA