defined in broad brushstrokes by studying cell morphology and the biogenesis of secretory proteins. Advances in the field have allowed a glimpse at the detailed mechanisms operating during various stages of this pathway. This review will describe the genetic and biochemical tools available for analyzing the mechanisms required for transport from the ER to the Golgi and our current level of understanding about the molecular machinery participating in this step of the secretory pathway .
Summary The cellular machinery responsible for conveying proteins between the endoplasmic reticulum and the Golgi is being investigated using genetics and biochemistry. A role for vesicles in mediating protein traffic between the ER and the Golgi has been established by characterizing yeast mutants defective in this process, and by using recently developed cell-free assays that measure ER to Golgi transport. These tools have also allowed the identification of several proteins crucial to intracellular protein trafficking. The characterization and possible functions of several GTP-binding proteins, peripheral membrane proteins, and an integral membrane protein during ER to Golgi transport are discussed here. Introduction Eukaryotic cells efficiently transport macromolecules to the cell surface, to the extracellular environment, and to assorted intracellular organelles. Proteins traveling through this transport system initiate their journey upon translocation through a lipid bilayer into the endoplasmic reticulum (ER). Unless destined to remain in the ER, transported proteins move to the Golgi apparatus where they travel sequentially through the cis, medial, and trans Golgi cisternae and upon arrival in the trans Golgi are packaged into secretory vesicles for release at the cell surface or targeted for transport to other intracellular organelles. Transport from the E R to the Golgi is the first step in this pathway requiring the transfer of secretory proteins between membranebounded organelles. In lower eukaryotes secretion is a constitutive process, rapid and efficient, though in mammalian cells with specialized secretory functions it may be more complicated. ER to Golgi transport is thought to be mediated by small vesicles, packed with secretory proteins, that bud from the reticular network of the E R and then recognize and fuse with the cis Golgi. It appears that the molecular operations involved in E R to Golgi vesicle budding, target recognition, and fusion may provide a ‘barebones’ paradigm for other transport phenomena such as travel between Golgi subcompartments, transport mediated by late secretory vesicles and granules, and endocytosis. Initially the steps of the secretory pathway were
ER to Golgi Transport Mutants Temperature-sensitive mutants that are defective in various stages of the secretory pathway have been isolated in the yeast Saccharomyces cerevisiae. The sect mutants (secretion) were isolated by the selection of mutagenized cells on a density gradient, as it had been observed that a block in secretion in Saccharomyces cerevisiae causes cells to become significantly denser than normal. From a sample of the densest mutagenized cells, mutants were isolated and twenty-three complementation groups identified amongst those mutants that accumulated active secretory proteins within the cell(’). Identification of the bet mutants (blocked early in transport) relied on the observation that a block in the transport of proteins prior to the Golgi prevents the addition of long mannose chains to glycoproteins. An early block in the secretory pathway should greatly diminish the incorporation of mannose into cellular protein; hence these mutants were identified by a suicide selection that required mutagenized cells to survive treatment with [3H]mannose(2). Ten see mutants (secl2, secl3, secl6, secl7, sec18, secl9, sec20, sec21, sec22, and sec23) and two bet mutants (bet1 and bet2) are unable to transport proteins from the ER to the Golgi and therefore accumulate vacuolar, plasma membrane, and extracellular proteins in the E R upon incubation at the nonpermissive temperature. Morphological analysis of these mutant cells with the electron microscope reveals they also accumulate an extensive network of E R membrane under nonpermissive conditions(’32). Y P T l is another yeast gene implicated in the secretory process. It was identified by virtue of its chromosomal location between the genes encoding actin and tubulin, and became the object of intense study when the sequence of its DNA showed it to be a member of the ras family of GTPbinding proteins. Conditional yptl mutants incubated at the restrictive temperature accumulate the secretory protein invertase in an immature, intracellular form and show proliferation of membranes and vesicles, characteristics consistent with cellular defects in ER to Golgi transport and transport between Golgi ~ i s t e r n a e ~ ~ , ~ ) . In addition to the Yptl protein, three of the proteins t The nomenclature rules for yeast genes and gene products are as followb. 1) Wild-type alleles of genes are denoted in upper case italics. 2) Mutant alleles of genes are denoted in lower case italics. 3) Names of proteins are represented in roman letters as proper names.
Table 1. Molecules proposed to frrnction in ER to Golgi transport A ) Proteins protein
MW
location and characteristics
Secl2p Sarlp Sec23p
70 kD 21 kD 85 kD
NSF=Secl8p
82-84 kD
YPtlP
23 kD
integral membrane glycoprotein ras-like GTP-binding protein peripheral membrane protein part of a 300-400 kD complex peripheral membrane/ cytosolic protein ras-like GTP-binding protein
as many as nine uncharacterized SEC and BET gene products
B) Small molecules ATP GTP Ca2+
encoded by SEC genes have been analyzed at the molecular level. The Secl2 protein is an integral membrane glycoprotein of approximately 70 kD(5). The protein encoded by SEC18 is a cytoplasmic 82-84 kD protein that exists in the cell in two forms, one soluble and one membrane-associated(@. Finally, the 84 kD Sec23 protein functions as part of a large complex (-400 kD) that is loosely associated with the cytosolic surface of an intracellular membrane(7) (Table 1). It is more difficult to identify the proteins that function as part of the secretory pathway in invertebrate and mammalian cells as these organisms are not readily amenable to the isolation of secretion mutants. Mammalian cells have been used to study some aspects of protein transport by analyzing cargo proteins that traverse the pathway during their biogenesis. The mechanisms involved in the retention of resident and incompletely folded proteins in the E R (reviewed in reference 9) have been studied in tissue culture cells by introducing mutant secretory proteins engineered in v i m . Mutational analyses of transmembrane viral proteins have identified E R retention signals in the Nterminus of the VP7 protein of the rotavirus SAll and in the cytoplasmic domain of the adenovirus E l 9 glycoprotein. A more general E R retention sequence, KDEL, has been identified on the C-terminus of a set of lumenal E R proteins exemplified by BiP (binding protein). Deletion of this sequence from BiP expressed in COS cells causes the protein to be exported through the secretory pathway, though secretion is slow, and addition of KDEL to the C-terminus of secretory and lysosomal proteins results in enhanced accumulation of these proteins in the ER(9). Study of KDEL fusions to the lysosomal protein cathepsin D has suggested the existence of a pathway for the recycling of exported proteins back to the E R . Though cathepsin D-KDEL is found in the ER it also carries mannose-linked phosphate, a modification thought to occur in a post E R compartment at or close to the cis Golgi. This observation has led to the hypothesis that E R proteins may not be actively retained within the ER but may be selectively recycled
back from the cis Golgi o r an intermediate compartment(’). Recent experiments with brefeldin A , a drug that inhibits E R to Golgi transport and causes the redistribution of cis and medial Golgi proteins to the ER, have also been interpreted as evidence for a Golgi to E R recycling mechanism, though abnormal fusion of organelles in the resence of the drug may also explain these results(’O.ll .
P
Reconstitution of ER to Golgi Transport In Vifro Cell-free assays of E R to Golgi transport have been developed using components from yeast or from mammalian cells. Both assays measure the acquisition of specific carbohydrate modifications by a representative secretory protein as an indication of its transfer from the E R to the Golgi. In vivo and in vitro, secretory proteins undergo posttranslational carbohydrate modifications that are hallmarks of their arrival at specific stages of the secretory pathway. Core-carbohydrate structures (composed of N-acetylglucosamine, glucose, and mannose) are transferred to an asparagine amino group on proteins in the E R and these structures are then trimmed and extended by the addition of outer chain carbohydrate in the Golgi complex(’*). Reconstitution of transport in Chinese hamster ovary (CHO) ‘semi-intact’ cells is followed by measuring the transport of radiolabeled vesicular stomatitis virus glycoprotein (VSV-G) from the E R to the Golgi of VSVinfected cells(13). A temperature-sensitive mutation in VSV-G inhibits the transport of the mutant protein out of the E R , resulting in the accumulation of mutant VSV-G exclusively in the E R of cells infected at the nonpermisssive temperature. In the E R the protein is modified by the addition of mannose-rich core-carbohydrate structures. When extracts from the infected cells are incubated in vitro at the permissive temperature, the accumulated VSV-G is efficiently transported to the Golgi where it encounters a Golgi-specific mannosidase-trimming enzyme (see Fig. 1). Trimming by the Golgi mannosidase renders the protein sensitive to the enzyme endoglycosidase D; therefore, after treatment with endoglycosidase D, protein that has reached the Golgi can be easily distinguished from protein remaining in the E R by a shift in electrophoretic mobility. Transport in this system requires cytosol, an ATP energy source, and is de endent upon physiological concentrations of ca2+(I3*‘). An in vitro assay using yeast components measures the transport of a radiolabeled secretory protein (the mating pheromone precursor, prepro-cv-factor) that is synthesized in vitro and introduced into the E R of yeast lysates by posttranslational tran~location(’~”~). Protein that is transported to the Golgi acquires outer chain carbohydrate and is identified by immune precipitation with antiserum that uniquely binds this carbohydrate (Fig. 1). As in the mammalian system, E R to Golgi transport in yeast lysates requires cytosolic proteins and ATP, and the membrane component has been divided
P
Golgi Fig. 1. The reconstitution of ER to Golgi transport in vitro. (A) ER-Golgi transport in 'semi-intact' tissue culture cells. A temperature-sensitive mutant VSV-G protein is radiolabeled and accumulated in the ER of CHO cells at the nonpermissive temperature where it acquires N-linked mannose-rich carbohydrate ( A ) . These cells are permeabilized, washed and returned to the permissive temperature with the addition of required components. Transport of G protein to the Golgi is measured by the amount of protein that has had its core carbohydrate trimmed by mannosidases and is therefore sensitive to digestion with endoglycosidase D. (B) ER+Golgi transport in yeast lysates. Prepro-a-factor labeled with 35S-methionine acquires N-linked core carbohydrate structures in the ER ( A ) . ER containing pro-a-factor is incubated at 15-30°C upon which the protein is transported to the Golgi and is modified with outer chain carbohydrate (0).Labeled pro-a-factor containing outer chain carbohydrate is quantified by immunoprecipitation with antiserum raised against a,l+6 mannose.
into fractions required for either donor (ER) or acceptor (Golgi) activity. The existence of the conditional yeast sec mutants and an in vitro assay allows the biochemical analysis of transport in extracts deficient in a protein known to be required for transport in the living cell. Using components from the sec23 mutant it has been shown that ER to Golgi transport in vitro requires the Sec23 protein, demonstrating that the in vitro reaction is a faithful representation of transport in vivo. The conditional defect in cells carrying a sec23 mutation is reproduced in vitro. Transport in sec23 lysates is temperature-sensitive(15)and can be restored at the nonpermissive temperature by the addition of partially purified fractions of a soluble form of Sec23 protein to these reactions(7). Using this assay, functional Sec23 protein has been purified in the form of a complex containing at least one other protein. It is hoped that a similar approach can be used to purify other SEC gene products and to analyze the behavior of membrane components involved in the transport process. Proteins Required for Formation and Fusion of a Vesicle Intermediate Historically, cell biologists have favored the idea that protein transport, specifically E R to Golgi transport and transport between Golgi cisternae, is mediated by vesicles. An alternate hypothesis posits that secretory proteins traverse a fine tubular network connecting the E R with the Golgi. Until recently little direct evidence for a vesicle intermediate existed; however, transport vesicles have now been identified both biochemically and morphologically in several mammalian systems. An
E R to Golgi transport intermediate consisting of small vesicles, pre-Golgi vacuoles, and tubular elements has been described in tissue culture cells. Virus-infected cells incubated at low temperature (15°C) accumulate viral membrane proteins in this morphologically heterogeneous intermediate, and protein accumulated within these elements progresses throu h the secretory pathway upon return of cells to 30°C(6). In addition, further transport in vitro of protein blocked at the intermediate requires cytosol and ATP(18). In other work, 60nm vesicles that bud from rat liver E R in the presence of ATP have been isolated by preparative free-flow electrophoresis. These vesicles have been shown to fuse with Golgi membranes immobilized on nitrocellulose in an event that is dependent on temperature but independent of ATP and cytosolic proteins("). Biochemical identification of E R to Golgi transport vesicles containing radiolabeled core-glycosylated secretory proteins from human hepatoma cells has also been reported, though the significance of these vesicles in vivo is unknown(20). Convincing support for a vesicular transport intermediate in yeast comes from recent morphological analysis of the yeast sec mutants and from analogies drawn between E R to Golgi transport and transport occurring between Golgi subcompartments. All of the sec mutants deficient in ER to Golgi transport demonstrate a proliferation of tubular ER-type membrane upon incubation at the restrictive temperature. However, closer inspection in the electron microscope of cells stained with KMn04, a reagent that highlights membrane structures, reveals a subclass of these mutants, secl7, secl8, and sec22, that also accumulate a significant number of 50 nm vesicles. The 50 nm vesicles
are believed to be true E R to Golgi transport intermediates because accumulation is diminished in mutant strains treated with a protein synthesis inhibitor, cycloheximide, and they do not accumulate in secZ7, secZ8, or sec22 cells if the cell also carries a sec mutation from the non-accumulating class, including secZ2, secl3, secl6, and sec23. For example, secl2secl8 cells do not accumulate vesicles at the nonpermissive temperature, indicating that the Secl2 protein acts before the Secl8 protein in a linear pathway of events@).These results establish that ongoing protein synthesis and SEC gene products are required for vesicle formation. It is therefore unlikely that the vesicles are artifacts of membrane fragmentation. Genetic evidence suggests the occurrence of protein interactions within each of the two classes of SEC gene products defined by the vesicle accumulation phenotype. A haploid yeast cell carrying temperature-sensitive mutations in both the SEC18 and SECZ7 genes is dead even at 2 4 T , a permissive temperature for the sec mutants, whereas a cell carrying the secZ8 mutation alone or in combination with any other E R to Golgi sec mutant grows normally at 24°C. This phenomenon is known as synthetic lethality@).secl8 and secl7 interact genetically and they both accumulate 50 nm vesicles. Genetic interaction is observed between secZ3, sec23, secZ6, and secZ2; mutations in these genes do not cause vesicle accumulation. As shown in Fig. 2, these data correlate and divide the transport event into two distinct steps. Perhaps the first step involves proteins required for budding of a vesicle from the E R and the second step involves targeting and fusion to the cis Golgi. Transport vesicles with a diameter of 75nm have
Transport vesicles SEC72’
13 \ 23
I
‘16/
Nucleus
S E C 2 11
U
OO
*oo
Golgi
SEC17-18
SEC22
0
SEC 19, S E C P O , BET?, B E T Z , V P T I
w
Fig. 2. Yeast genes required for protein transport from the ER to the Golgi. Genetic data imply an interaction between the SEC17 and SEC18 gene products, which along with the SEC22 gene product, appear to be required for fusion of transport vesicles with the Golgi. The Secl2, 13, 16, and 23 proteins are believed to be required for transport vesicle formation. Genetic interactions among the secl2, secZ3, secl6, and sec23 mutants are indicated by connecting bars. The SECl9, SEC20, B E T I , BET2, and Y P T l gene products have not yet been classified in terms of their role in vesicle formation or vesicle fusion.
been identified in lysates of mammalian cells as the intermediate in transport between subcompartments of the Golgi. In vitro these vesicles are observed to bud from Golgi membranes, travel to a target Golgi cisternae, and then fuse with the target membrane(21). Several inhibitors of intraGolgi transport in vitro cause accumulation of the vesicles and have allowed their purification. An in vitro assay for transport between Golgi cisternae has identified several proteins that act together in promoting vesicle One of these, NEM-sensitive fusion protein (NSF), is the mammalian homologue of the Secl8 protein; the two amino acid sequences are 48% identical(24). In addition, NSF is required for E R to Golgi transport reconstituted in CHO semi-intact cells(25).Inactivation of NSF causes the accumulation of vesicles attached to their target compartment but unable to fuse with the membrane. The homology between the Secl8 protein and NSF, a protein identified because it is required for fusion of small transport vesicles with their target membrane, provides a strong link between the transport vesicles observed to accumulate in secl8 mutants in vivo and those identified in vitro as intraGolgi transport intermediates. In addition to proteins required for fusion of membranes, others will be necessary for recognition between the vesicle and its target, perhaps a receptor protein in the Golgi will detect a vesicular protein exposing the correct ‘address’. A correct match between address and receptor may then trigger a structural change on the vesicle, making it competent for fusion. The accumulation of vesicles in the secl7 and sec22 mutants implies that the Secl7 and Sec22 proteins along with others will be required for these functions. The gene products of SEC23, SECZ6, SECZS, and SECZ2 are required for vesicle formation and therefore may act in the assembly and pinching off of transport vesicles from the E R membrane. The Sec23 protein functions in the form of a large complex peripherally attached to the surface of the membrane. The role of this complex in vesicle morphogenesis is as yet undefined, though potential functions include a role in recognition of areas of membrane competent to bud, a role in the mechanical action of pinching off the bud from the ER, and/or a structural role as a coat for ER to Golgi transport vesicles. Clathrin, a protein that forms a basket-like structure or coat around vesicles budding from the trans Golgi and vesicles formed during receptor-mediated endocytosis, is unlikely to be involved in early steps of the secretory pathway as yeast mutants deficient in clathrin heavy chain, though quite sickly, transport and localize secretory proteins normally(26). However, the vesicles isolated as intraGolgi transport intermediates appear to carry a coat-like structure (this structure does not react with clathrin antiserum)(21),and such a coat may also be required on E R to Golgi transport vesicles. It is also possible that a peripheral membrane complex required for transport may associate with the
cytoskeleton. Microtubule-based structures have been implicated in the biogenesis of the E R ( 2 7 ) but may not play a direct role in ER to Golgi transport, since conditional mutations in the yeast tubulin genes have little effect on the export of the secretory protein invertase(28).Yeast actin mutations partially block secretion, but this block appears to be late in the pathway(29).Thus, there is no strong evidence for the participation of actin-based cytoskeleton in ER to Golgi transport. If transport vesicles do not travel along a known cytoskeletal framework from their origin to their destination, it is not clear how their movement between compartments is directed. Perhaps an as yet unidentified cytoskeletal protein or structure is required for ER to Golgi transport. Alternatively, the ER and the cis Golgi in living cells may be organized in such a way that diffusion of vesicles through the cytoplasm results in efficient transport. GTP-Binding Proteins in ER to Golgi Transport The observation that the yeast Sec4 protein, required for transport of secretory vesicles to the plasma membrane, is homologous to the ras-like family of GTPbinding proteins initiated the study of GTP-binding proteins in various stages of the secretory pathway(30). Several lines of evidence show that they are required for ER to Golgi transport. ER to Golgi transport in both CHO and yeast lysates is blocked by the nonh drolyzable GTP analogue, GTPyS, and by A1F4-(1431'16), which in the presence of GDP mimics the y-phosphate group of GTP. These reagents are well-established activators of the G proteins involved in signal transduction and also inhibit transport between Golgi compartments in In vivo, conditional mutants in the YPTZ yeast gene have a phenoty e consistent with a defect in ER to Golgi transport( f), and lysates prepared from yptl cells are unable to support ER to Golgi transport in v i t r ~ ( ~ l , The ~ ~ ) . 23 kD Yptl protein binds and hydrolyzes GTP and has been localized to the Golgi complex by immunofluorescence studies of yeast and mammalian cells(3). Another GTP-binding protein implicated in ER to Golgi transport in yeast is encoded by the SARl gene, which was isolated as a multicopy suppressor of the secl2 mutation. A two-fold elevation in the expression of SARl is sufficient to suppress the temperature-sensitive phenotype of secl2 mutants. Depletion of the 21 kD Sarl protein from yeast cells causes accumulation of core-glycosylated secretory proteins in the ER and the arrest of cell There may be other GTP-binding proteins required for ER to Golgi transport that govern key regulatory steps of the process. Such regulatory steps might be the determination of vesicle cargo, vesicle size, and the correct recognition of the target membrane. The genetic interaction of SARl with SEC12 implies that it acts early, probably in vesicle formation. As to later events, work with CHO extracts has shown that a GTP-binding
protein is required after formation of the 15"C transport intermediate(14). Proteins highly homologous to Yptl protein have been identified in rat(34)and and a similar homologue may be a target of GTPyS in CHO cells. Several models have been proposed for the role of GTP-binding proteins in intracellular transport("). GTP is not likely to provide a major source of energy for the mechanical action necessary for vesicle transport. However, the energy carried by GTP in a highenergy complex may serve to promote fidelity of vesicle budding and targeting as the GTP-binding translation factor EfTu ensures the fidelity of translation of a nucleotide sequence into an amino acid sequence. GTP-binding proteins involved in transport may also function in a signal transduction pathway, mediating the transfer of information from the lumen of an organelle through an integral membrane protein to the cytosolic surface or vice versa. Signals transmitted across the ER membrane in this way could reflect the local concentration of soluble secretory proteins on the lumenal side of the membrane or the availability of structural components to form a vesicle coat on the cytosolic surface. Whatever their mechanism of action, the understanding of the role played by GTP-binding proteins should provide insight into the regulation of intracellular transport. Acknowledgements We thank Chris Kaiser, Nancy Pryer, and Sylvia Sanders for valuable comments on the manuscript. References 1 SCHEKMAN. R. (1985). Protein localization and membrane traffic in yeast. Annu. Rev. Cell B i d . 1, 115-143. 2 NEWMAN,A. P. AND FERRO-NOVICK, S . (1987). Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection. J . Cell Biol. 105, 1587-1594. 3 SEGEV,N . , MULHOLLAND, J. AND BOTSTEIN, D. (1988). The yeast GTPbinding YPTl protein and a mammalian counterpart are associated with the secretion machinery. Cell 52, 915-924. 4 SCHMITT, H. D., PUZICHA.M. AND GALLWIIZ. D. (1988). Study of a temperature-sensitive mutant of the ms-related YPTl gene product in yeast suggests a role in the regulation of intracellular calcium. Cell 53, 635-647. 5 NAKANO.A , , BRADA, D. AND SCHBKMAN, R. (1988). A membrane glycoprotein, SeclZp, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast. J. Cell B i d . 107, 851-863. 6 EAKLE,K. A . , BERNSTEIN, M . AND EMR,S. D. (1988). Characterization of a component of the yeast secretion machinery: identification of the SECIN gcnc product. Mol. Cell. B i d . 8, 4098-4109. 7 HICKE,L. A N D SCHEKMAN. R. (1989). Yeast Sec23p acts in the cytoplasm 1 0 promote protein transport from the ER to the Golgi complex in vivo and in vitro. EMBO J. 8, 1677-1684. 8 KAISER,C. AND SCHEKMAN, R, (1990). Cell, in press. 9 PELHAM, H. R . B. (1989). Control of protein exit from the endoplasmic reticulum. Annu. Rev. Cell Biol. 5 , 1-23. 10 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, 841-813. 11 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. 12 KORNFELD, R. AND KORNFELD, S. (1985). Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 54, 631-634. 13 BECKERS, C. J . M., KELLER, D. S. AND BALCH,W . E. (1988). Semi-intact
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BALCH.W. E. (1989). Vesicular transport between the endoplasmic reticulum and the Golgi stack requires the NEM-sensitive fusion protein. Nafure 339, 397-398. 26 PAYNE,G. S. AND SCHEKMAN, R. (1985). A test of clathrin function in protein secretion and cell growth. Science 230, 109-1014. 27 DABORA, S. L. AND SHEETZ,M. P. (1988). The microtubule-dependent formation of a tubulovesicular network with characteristics of the ER from cultured cell extracts. Cell 54, 27-35. 28 HUFFAKER, T. C., THOMAS, J . H. A N D BOTSTEIN, D. (1988). Diverse effects of Ptubulin mutations on microtubule formation and function. J . C d . B i d . 106, 1997-2010. 29 NOVICK, P. AND BOTSTEIN, D. (1985). Phenotypic analysis of temperaturesensitive yeast actin mutants. Cell 40,405-416. 30 BOURNE, H. R. (1988). D o GTPases direct membrane traffic in becretion? Cell53,669-671. 31 BACON,R. A,, SALMINEN, A,, RUOHOLA, H., NOVICK. P. A N D FERRONOVICK. S . (1989). The GTP-binding protein Yptl is required for transport in v i m : the Golgi apparatus is defective in y p f l mutants. J . CeN Biol. 109, 1015-1022. 32 BAKER, D., WUESTEHUBE, L., SCHEKMAN, R., BOTSTEIN, D. A N D SEGEV, N. (1989). The GTP-binding Yptl prorein and Ca2+ function independently in a cell-free protein transport reaction. Proc. Nufl Arud. Sci. USA 87, 355-359. 33 NAKANO, A. AND MURAMATSU, M. (1989). A novel GTP-binding protein, Sarlp, is involved in transport from the endoplasmic reticulum to the Golgi apparatus. J. Cell Biol. 109, 2677-2691. 34 TOUCHOT, N., CHARDIN, P. A N D TAVITIAN, A. (1987). Four additional members of the rus gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library. Proc. Nafl Arad. Sci. USA, 84, 8210-8214. 35 HAUBRUCK, H., DISELA,c.,WAGNER, P. AND GALLWITZ, D. (1987). The ros related ypf protein is an ubiquitous eukaryotic protein: isolation and sequence analysis of mouse cDNA clones highly homologous to the yeast YPTl gene. E M B O J. 6, 4049-4053.
Linda Hicke and Randy Schekman are at the Division of Biochemistry and Molecular Biology, Barker Hall, University of California, Berkeley, CA 94720, USA.
Summary The cellular machinery responsible for conveying proteins between the endoplasmic reticulum and the Golgi is being investigated using genetics and biochemistry. A role for vesicles in mediating protein traffic between the ER and the Golgi has been established by characterizing yeast mutants defective in this process, and by using recently developed cell-free assays that measure ER to Golgi transport. These tools have also allowed the identification of several proteins crucial to intracellular protein trafficking. The characterization and possible functions of several GTP-binding proteins, peripheral membrane proteins, and an integral membrane protein during ER to Golgi transport are discussed here. Introduction Eukaryotic cells efficiently transport macromolecules to the cell surface, to the extracellular environment, and to assorted intracellular organelles. Proteins traveling through this transport system initiate their journey upon translocation through a lipid bilayer into the endoplasmic reticulum (ER). Unless destined to remain in the ER, transported proteins move to the Golgi apparatus where they travel sequentially through the cis, medial, and trans Golgi cisternae and upon arrival in the trans Golgi are packaged into secretory vesicles for release at the cell surface or targeted for transport to other intracellular organelles. Transport from the E R to the Golgi is the first step in this pathway requiring the transfer of secretory proteins between membranebounded organelles. In lower eukaryotes secretion is a constitutive process, rapid and efficient, though in mammalian cells with specialized secretory functions it may be more complicated. ER to Golgi transport is thought to be mediated by small vesicles, packed with secretory proteins, that bud from the reticular network of the E R and then recognize and fuse with the cis Golgi. It appears that the molecular operations involved in E R to Golgi vesicle budding, target recognition, and fusion may provide a ‘barebones’ paradigm for other transport phenomena such as travel between Golgi subcompartments, transport mediated by late secretory vesicles and granules, and endocytosis. Initially the steps of the secretory pathway were
ER to Golgi Transport Mutants Temperature-sensitive mutants that are defective in various stages of the secretory pathway have been isolated in the yeast Saccharomyces cerevisiae. The sect mutants (secretion) were isolated by the selection of mutagenized cells on a density gradient, as it had been observed that a block in secretion in Saccharomyces cerevisiae causes cells to become significantly denser than normal. From a sample of the densest mutagenized cells, mutants were isolated and twenty-three complementation groups identified amongst those mutants that accumulated active secretory proteins within the cell(’). Identification of the bet mutants (blocked early in transport) relied on the observation that a block in the transport of proteins prior to the Golgi prevents the addition of long mannose chains to glycoproteins. An early block in the secretory pathway should greatly diminish the incorporation of mannose into cellular protein; hence these mutants were identified by a suicide selection that required mutagenized cells to survive treatment with [3H]mannose(2). Ten see mutants (secl2, secl3, secl6, secl7, sec18, secl9, sec20, sec21, sec22, and sec23) and two bet mutants (bet1 and bet2) are unable to transport proteins from the ER to the Golgi and therefore accumulate vacuolar, plasma membrane, and extracellular proteins in the E R upon incubation at the nonpermissive temperature. Morphological analysis of these mutant cells with the electron microscope reveals they also accumulate an extensive network of E R membrane under nonpermissive conditions(’32). Y P T l is another yeast gene implicated in the secretory process. It was identified by virtue of its chromosomal location between the genes encoding actin and tubulin, and became the object of intense study when the sequence of its DNA showed it to be a member of the ras family of GTPbinding proteins. Conditional yptl mutants incubated at the restrictive temperature accumulate the secretory protein invertase in an immature, intracellular form and show proliferation of membranes and vesicles, characteristics consistent with cellular defects in ER to Golgi transport and transport between Golgi ~ i s t e r n a e ~ ~ , ~ ) . In addition to the Yptl protein, three of the proteins t The nomenclature rules for yeast genes and gene products are as followb. 1) Wild-type alleles of genes are denoted in upper case italics. 2) Mutant alleles of genes are denoted in lower case italics. 3) Names of proteins are represented in roman letters as proper names.
Table 1. Molecules proposed to frrnction in ER to Golgi transport A ) Proteins protein
MW
location and characteristics
Secl2p Sarlp Sec23p
70 kD 21 kD 85 kD
NSF=Secl8p
82-84 kD
YPtlP
23 kD
integral membrane glycoprotein ras-like GTP-binding protein peripheral membrane protein part of a 300-400 kD complex peripheral membrane/ cytosolic protein ras-like GTP-binding protein
as many as nine uncharacterized SEC and BET gene products
B) Small molecules ATP GTP Ca2+
encoded by SEC genes have been analyzed at the molecular level. The Secl2 protein is an integral membrane glycoprotein of approximately 70 kD(5). The protein encoded by SEC18 is a cytoplasmic 82-84 kD protein that exists in the cell in two forms, one soluble and one membrane-associated(@. Finally, the 84 kD Sec23 protein functions as part of a large complex (-400 kD) that is loosely associated with the cytosolic surface of an intracellular membrane(7) (Table 1). It is more difficult to identify the proteins that function as part of the secretory pathway in invertebrate and mammalian cells as these organisms are not readily amenable to the isolation of secretion mutants. Mammalian cells have been used to study some aspects of protein transport by analyzing cargo proteins that traverse the pathway during their biogenesis. The mechanisms involved in the retention of resident and incompletely folded proteins in the E R (reviewed in reference 9) have been studied in tissue culture cells by introducing mutant secretory proteins engineered in v i m . Mutational analyses of transmembrane viral proteins have identified E R retention signals in the Nterminus of the VP7 protein of the rotavirus SAll and in the cytoplasmic domain of the adenovirus E l 9 glycoprotein. A more general E R retention sequence, KDEL, has been identified on the C-terminus of a set of lumenal E R proteins exemplified by BiP (binding protein). Deletion of this sequence from BiP expressed in COS cells causes the protein to be exported through the secretory pathway, though secretion is slow, and addition of KDEL to the C-terminus of secretory and lysosomal proteins results in enhanced accumulation of these proteins in the ER(9). Study of KDEL fusions to the lysosomal protein cathepsin D has suggested the existence of a pathway for the recycling of exported proteins back to the E R . Though cathepsin D-KDEL is found in the ER it also carries mannose-linked phosphate, a modification thought to occur in a post E R compartment at or close to the cis Golgi. This observation has led to the hypothesis that E R proteins may not be actively retained within the ER but may be selectively recycled
back from the cis Golgi o r an intermediate compartment(’). Recent experiments with brefeldin A , a drug that inhibits E R to Golgi transport and causes the redistribution of cis and medial Golgi proteins to the ER, have also been interpreted as evidence for a Golgi to E R recycling mechanism, though abnormal fusion of organelles in the resence of the drug may also explain these results(’O.ll .
P
Reconstitution of ER to Golgi Transport In Vifro Cell-free assays of E R to Golgi transport have been developed using components from yeast or from mammalian cells. Both assays measure the acquisition of specific carbohydrate modifications by a representative secretory protein as an indication of its transfer from the E R to the Golgi. In vivo and in vitro, secretory proteins undergo posttranslational carbohydrate modifications that are hallmarks of their arrival at specific stages of the secretory pathway. Core-carbohydrate structures (composed of N-acetylglucosamine, glucose, and mannose) are transferred to an asparagine amino group on proteins in the E R and these structures are then trimmed and extended by the addition of outer chain carbohydrate in the Golgi complex(’*). Reconstitution of transport in Chinese hamster ovary (CHO) ‘semi-intact’ cells is followed by measuring the transport of radiolabeled vesicular stomatitis virus glycoprotein (VSV-G) from the E R to the Golgi of VSVinfected cells(13). A temperature-sensitive mutation in VSV-G inhibits the transport of the mutant protein out of the E R , resulting in the accumulation of mutant VSV-G exclusively in the E R of cells infected at the nonpermisssive temperature. In the E R the protein is modified by the addition of mannose-rich core-carbohydrate structures. When extracts from the infected cells are incubated in vitro at the permissive temperature, the accumulated VSV-G is efficiently transported to the Golgi where it encounters a Golgi-specific mannosidase-trimming enzyme (see Fig. 1). Trimming by the Golgi mannosidase renders the protein sensitive to the enzyme endoglycosidase D; therefore, after treatment with endoglycosidase D, protein that has reached the Golgi can be easily distinguished from protein remaining in the E R by a shift in electrophoretic mobility. Transport in this system requires cytosol, an ATP energy source, and is de endent upon physiological concentrations of ca2+(I3*‘). An in vitro assay using yeast components measures the transport of a radiolabeled secretory protein (the mating pheromone precursor, prepro-cv-factor) that is synthesized in vitro and introduced into the E R of yeast lysates by posttranslational tran~location(’~”~). Protein that is transported to the Golgi acquires outer chain carbohydrate and is identified by immune precipitation with antiserum that uniquely binds this carbohydrate (Fig. 1). As in the mammalian system, E R to Golgi transport in yeast lysates requires cytosolic proteins and ATP, and the membrane component has been divided
P
Golgi Fig. 1. The reconstitution of ER to Golgi transport in vitro. (A) ER-Golgi transport in 'semi-intact' tissue culture cells. A temperature-sensitive mutant VSV-G protein is radiolabeled and accumulated in the ER of CHO cells at the nonpermissive temperature where it acquires N-linked mannose-rich carbohydrate ( A ) . These cells are permeabilized, washed and returned to the permissive temperature with the addition of required components. Transport of G protein to the Golgi is measured by the amount of protein that has had its core carbohydrate trimmed by mannosidases and is therefore sensitive to digestion with endoglycosidase D. (B) ER+Golgi transport in yeast lysates. Prepro-a-factor labeled with 35S-methionine acquires N-linked core carbohydrate structures in the ER ( A ) . ER containing pro-a-factor is incubated at 15-30°C upon which the protein is transported to the Golgi and is modified with outer chain carbohydrate (0).Labeled pro-a-factor containing outer chain carbohydrate is quantified by immunoprecipitation with antiserum raised against a,l+6 mannose.
into fractions required for either donor (ER) or acceptor (Golgi) activity. The existence of the conditional yeast sec mutants and an in vitro assay allows the biochemical analysis of transport in extracts deficient in a protein known to be required for transport in the living cell. Using components from the sec23 mutant it has been shown that ER to Golgi transport in vitro requires the Sec23 protein, demonstrating that the in vitro reaction is a faithful representation of transport in vivo. The conditional defect in cells carrying a sec23 mutation is reproduced in vitro. Transport in sec23 lysates is temperature-sensitive(15)and can be restored at the nonpermissive temperature by the addition of partially purified fractions of a soluble form of Sec23 protein to these reactions(7). Using this assay, functional Sec23 protein has been purified in the form of a complex containing at least one other protein. It is hoped that a similar approach can be used to purify other SEC gene products and to analyze the behavior of membrane components involved in the transport process. Proteins Required for Formation and Fusion of a Vesicle Intermediate Historically, cell biologists have favored the idea that protein transport, specifically E R to Golgi transport and transport between Golgi cisternae, is mediated by vesicles. An alternate hypothesis posits that secretory proteins traverse a fine tubular network connecting the E R with the Golgi. Until recently little direct evidence for a vesicle intermediate existed; however, transport vesicles have now been identified both biochemically and morphologically in several mammalian systems. An
E R to Golgi transport intermediate consisting of small vesicles, pre-Golgi vacuoles, and tubular elements has been described in tissue culture cells. Virus-infected cells incubated at low temperature (15°C) accumulate viral membrane proteins in this morphologically heterogeneous intermediate, and protein accumulated within these elements progresses throu h the secretory pathway upon return of cells to 30°C(6). In addition, further transport in vitro of protein blocked at the intermediate requires cytosol and ATP(18). In other work, 60nm vesicles that bud from rat liver E R in the presence of ATP have been isolated by preparative free-flow electrophoresis. These vesicles have been shown to fuse with Golgi membranes immobilized on nitrocellulose in an event that is dependent on temperature but independent of ATP and cytosolic proteins("). Biochemical identification of E R to Golgi transport vesicles containing radiolabeled core-glycosylated secretory proteins from human hepatoma cells has also been reported, though the significance of these vesicles in vivo is unknown(20). Convincing support for a vesicular transport intermediate in yeast comes from recent morphological analysis of the yeast sec mutants and from analogies drawn between E R to Golgi transport and transport occurring between Golgi subcompartments. All of the sec mutants deficient in ER to Golgi transport demonstrate a proliferation of tubular ER-type membrane upon incubation at the restrictive temperature. However, closer inspection in the electron microscope of cells stained with KMn04, a reagent that highlights membrane structures, reveals a subclass of these mutants, secl7, secl8, and sec22, that also accumulate a significant number of 50 nm vesicles. The 50 nm vesicles
are believed to be true E R to Golgi transport intermediates because accumulation is diminished in mutant strains treated with a protein synthesis inhibitor, cycloheximide, and they do not accumulate in secZ7, secZ8, or sec22 cells if the cell also carries a sec mutation from the non-accumulating class, including secZ2, secl3, secl6, and sec23. For example, secl2secl8 cells do not accumulate vesicles at the nonpermissive temperature, indicating that the Secl2 protein acts before the Secl8 protein in a linear pathway of events@).These results establish that ongoing protein synthesis and SEC gene products are required for vesicle formation. It is therefore unlikely that the vesicles are artifacts of membrane fragmentation. Genetic evidence suggests the occurrence of protein interactions within each of the two classes of SEC gene products defined by the vesicle accumulation phenotype. A haploid yeast cell carrying temperature-sensitive mutations in both the SEC18 and SECZ7 genes is dead even at 2 4 T , a permissive temperature for the sec mutants, whereas a cell carrying the secZ8 mutation alone or in combination with any other E R to Golgi sec mutant grows normally at 24°C. This phenomenon is known as synthetic lethality@).secl8 and secl7 interact genetically and they both accumulate 50 nm vesicles. Genetic interaction is observed between secZ3, sec23, secZ6, and secZ2; mutations in these genes do not cause vesicle accumulation. As shown in Fig. 2, these data correlate and divide the transport event into two distinct steps. Perhaps the first step involves proteins required for budding of a vesicle from the E R and the second step involves targeting and fusion to the cis Golgi. Transport vesicles with a diameter of 75nm have
Transport vesicles SEC72’
13 \ 23
I
‘16/
Nucleus
S E C 2 11
U
OO
*oo
Golgi
SEC17-18
SEC22
0
SEC 19, S E C P O , BET?, B E T Z , V P T I
w
Fig. 2. Yeast genes required for protein transport from the ER to the Golgi. Genetic data imply an interaction between the SEC17 and SEC18 gene products, which along with the SEC22 gene product, appear to be required for fusion of transport vesicles with the Golgi. The Secl2, 13, 16, and 23 proteins are believed to be required for transport vesicle formation. Genetic interactions among the secl2, secZ3, secl6, and sec23 mutants are indicated by connecting bars. The SECl9, SEC20, B E T I , BET2, and Y P T l gene products have not yet been classified in terms of their role in vesicle formation or vesicle fusion.
been identified in lysates of mammalian cells as the intermediate in transport between subcompartments of the Golgi. In vitro these vesicles are observed to bud from Golgi membranes, travel to a target Golgi cisternae, and then fuse with the target membrane(21). Several inhibitors of intraGolgi transport in vitro cause accumulation of the vesicles and have allowed their purification. An in vitro assay for transport between Golgi cisternae has identified several proteins that act together in promoting vesicle One of these, NEM-sensitive fusion protein (NSF), is the mammalian homologue of the Secl8 protein; the two amino acid sequences are 48% identical(24). In addition, NSF is required for E R to Golgi transport reconstituted in CHO semi-intact cells(25).Inactivation of NSF causes the accumulation of vesicles attached to their target compartment but unable to fuse with the membrane. The homology between the Secl8 protein and NSF, a protein identified because it is required for fusion of small transport vesicles with their target membrane, provides a strong link between the transport vesicles observed to accumulate in secl8 mutants in vivo and those identified in vitro as intraGolgi transport intermediates. In addition to proteins required for fusion of membranes, others will be necessary for recognition between the vesicle and its target, perhaps a receptor protein in the Golgi will detect a vesicular protein exposing the correct ‘address’. A correct match between address and receptor may then trigger a structural change on the vesicle, making it competent for fusion. The accumulation of vesicles in the secl7 and sec22 mutants implies that the Secl7 and Sec22 proteins along with others will be required for these functions. The gene products of SEC23, SECZ6, SECZS, and SECZ2 are required for vesicle formation and therefore may act in the assembly and pinching off of transport vesicles from the E R membrane. The Sec23 protein functions in the form of a large complex peripherally attached to the surface of the membrane. The role of this complex in vesicle morphogenesis is as yet undefined, though potential functions include a role in recognition of areas of membrane competent to bud, a role in the mechanical action of pinching off the bud from the ER, and/or a structural role as a coat for ER to Golgi transport vesicles. Clathrin, a protein that forms a basket-like structure or coat around vesicles budding from the trans Golgi and vesicles formed during receptor-mediated endocytosis, is unlikely to be involved in early steps of the secretory pathway as yeast mutants deficient in clathrin heavy chain, though quite sickly, transport and localize secretory proteins normally(26). However, the vesicles isolated as intraGolgi transport intermediates appear to carry a coat-like structure (this structure does not react with clathrin antiserum)(21),and such a coat may also be required on E R to Golgi transport vesicles. It is also possible that a peripheral membrane complex required for transport may associate with the
cytoskeleton. Microtubule-based structures have been implicated in the biogenesis of the E R ( 2 7 ) but may not play a direct role in ER to Golgi transport, since conditional mutations in the yeast tubulin genes have little effect on the export of the secretory protein invertase(28).Yeast actin mutations partially block secretion, but this block appears to be late in the pathway(29).Thus, there is no strong evidence for the participation of actin-based cytoskeleton in ER to Golgi transport. If transport vesicles do not travel along a known cytoskeletal framework from their origin to their destination, it is not clear how their movement between compartments is directed. Perhaps an as yet unidentified cytoskeletal protein or structure is required for ER to Golgi transport. Alternatively, the ER and the cis Golgi in living cells may be organized in such a way that diffusion of vesicles through the cytoplasm results in efficient transport. GTP-Binding Proteins in ER to Golgi Transport The observation that the yeast Sec4 protein, required for transport of secretory vesicles to the plasma membrane, is homologous to the ras-like family of GTPbinding proteins initiated the study of GTP-binding proteins in various stages of the secretory pathway(30). Several lines of evidence show that they are required for ER to Golgi transport. ER to Golgi transport in both CHO and yeast lysates is blocked by the nonh drolyzable GTP analogue, GTPyS, and by A1F4-(1431'16), which in the presence of GDP mimics the y-phosphate group of GTP. These reagents are well-established activators of the G proteins involved in signal transduction and also inhibit transport between Golgi compartments in In vivo, conditional mutants in the YPTZ yeast gene have a phenoty e consistent with a defect in ER to Golgi transport( f), and lysates prepared from yptl cells are unable to support ER to Golgi transport in v i t r ~ ( ~ l , The ~ ~ ) . 23 kD Yptl protein binds and hydrolyzes GTP and has been localized to the Golgi complex by immunofluorescence studies of yeast and mammalian cells(3). Another GTP-binding protein implicated in ER to Golgi transport in yeast is encoded by the SARl gene, which was isolated as a multicopy suppressor of the secl2 mutation. A two-fold elevation in the expression of SARl is sufficient to suppress the temperature-sensitive phenotype of secl2 mutants. Depletion of the 21 kD Sarl protein from yeast cells causes accumulation of core-glycosylated secretory proteins in the ER and the arrest of cell There may be other GTP-binding proteins required for ER to Golgi transport that govern key regulatory steps of the process. Such regulatory steps might be the determination of vesicle cargo, vesicle size, and the correct recognition of the target membrane. The genetic interaction of SARl with SEC12 implies that it acts early, probably in vesicle formation. As to later events, work with CHO extracts has shown that a GTP-binding
protein is required after formation of the 15"C transport intermediate(14). Proteins highly homologous to Yptl protein have been identified in rat(34)and and a similar homologue may be a target of GTPyS in CHO cells. Several models have been proposed for the role of GTP-binding proteins in intracellular transport("). GTP is not likely to provide a major source of energy for the mechanical action necessary for vesicle transport. However, the energy carried by GTP in a highenergy complex may serve to promote fidelity of vesicle budding and targeting as the GTP-binding translation factor EfTu ensures the fidelity of translation of a nucleotide sequence into an amino acid sequence. GTP-binding proteins involved in transport may also function in a signal transduction pathway, mediating the transfer of information from the lumen of an organelle through an integral membrane protein to the cytosolic surface or vice versa. Signals transmitted across the ER membrane in this way could reflect the local concentration of soluble secretory proteins on the lumenal side of the membrane or the availability of structural components to form a vesicle coat on the cytosolic surface. Whatever their mechanism of action, the understanding of the role played by GTP-binding proteins should provide insight into the regulation of intracellular transport. Acknowledgements We thank Chris Kaiser, Nancy Pryer, and Sylvia Sanders for valuable comments on the manuscript. References 1 SCHEKMAN. R. (1985). Protein localization and membrane traffic in yeast. Annu. Rev. Cell B i d . 1, 115-143. 2 NEWMAN,A. P. AND FERRO-NOVICK, S . (1987). Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection. J . Cell Biol. 105, 1587-1594. 3 SEGEV,N . , MULHOLLAND, J. AND BOTSTEIN, D. (1988). The yeast GTPbinding YPTl protein and a mammalian counterpart are associated with the secretion machinery. Cell 52, 915-924. 4 SCHMITT, H. D., PUZICHA.M. AND GALLWIIZ. D. (1988). Study of a temperature-sensitive mutant of the ms-related YPTl gene product in yeast suggests a role in the regulation of intracellular calcium. Cell 53, 635-647. 5 NAKANO.A , , BRADA, D. AND SCHBKMAN, R. (1988). A membrane glycoprotein, SeclZp, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast. J. Cell B i d . 107, 851-863. 6 EAKLE,K. A . , BERNSTEIN, M . AND EMR,S. D. (1988). Characterization of a component of the yeast secretion machinery: identification of the SECIN gcnc product. Mol. Cell. B i d . 8, 4098-4109. 7 HICKE,L. A N D SCHEKMAN. R. (1989). Yeast Sec23p acts in the cytoplasm 1 0 promote protein transport from the ER to the Golgi complex in vivo and in vitro. EMBO J. 8, 1677-1684. 8 KAISER,C. AND SCHEKMAN, R, (1990). Cell, in press. 9 PELHAM, H. R . B. (1989). Control of protein exit from the endoplasmic reticulum. Annu. Rev. Cell Biol. 5 , 1-23. 10 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, 841-813. 11 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. 12 KORNFELD, R. AND KORNFELD, S. (1985). Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 54, 631-634. 13 BECKERS, C. J . M., KELLER, D. S. AND BALCH,W . E. (1988). Semi-intact
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Linda Hicke and Randy Schekman are at the Division of Biochemistry and Molecular Biology, Barker Hall, University of California, Berkeley, CA 94720, USA.
