Benjamin S. Glick

The Mechanisms of Vesicle Budding and Fusion

Genetic and biochemical analyses of the secretory pathway have produced a detailed picture of the molecular mechanisms involved in selective cargo transport between organelles. This transport occurs by means of vesicular intermediates that bud from a donor compartment and fuse with an acceptor compartment. Vesicle budding and cargo selection are mediated by protein coats, while vesicle targeting and fusion depend on a machinery that includes the SNARE proteins. Precise regulation of these two aspects of vesicular transport ensures efficient cargo transfer while preserving organelle identity.

Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed)

The red fluorescent protein DsRed has spectral properties that are ideal for dual-color experiments with green fluorescent protein (GFP). But wild-type DsRed has several drawbacks, including slow chromophore maturation and poor solubility. To overcome the slow maturation, we used random and directed mutagenesis to create DsRed variants that mature 10–15 times faster than the wild-type protein. An asparagine-to-glutamine substitution at position 42 greatly accelerates the maturation of DsRed, but also increases the level of green emission. Additional amino acid substitutions suppress this green emission while further accelerating the maturation. To enhance the solubility of DsRed, we reduced the net charge near the N terminus of the protein. The optimized DsRed variants yield bright fluorescence even in rapidly growing organisms such as yeast.

Golgi maturation visualized in living yeast.

The Golgi apparatus is composed of biochemically distinct early (cis, medial) and late (trans, TGN) cisternae. There is debate about the nature of these cisternae. The stable compartments model predicts that each cisterna is a long-lived structure that retains a characteristic set of Golgi-resident proteins. In this view, secretory cargo proteins are transported by vesicles from one cisterna to the next. The cisternal maturation model predicts that each cisterna is a transient structure that matures from early to late by acquiring and then losing specific Golgi-resident proteins. In this view, secretory cargo proteins traverse the Golgi by remaining within the maturing cisternae. Various observations have been interpreted as supporting one or the other mechanism. Here we provide a direct test of the two models using three-dimensional time-lapse fluorescence microscopy of the yeast Saccharomyces cerevisiae. This approach reveals that individual cisternae mature, and do so at a consistent rate. In parallel, we used pulse–chase analysis to measure the transport of two secretory cargo proteins. The rate of cisternal maturation matches the rate of protein transport through the secretory pathway, suggesting that cisternal maturation can account for the kinetics of secretory traffic.

Can Hsp70 proteins act as force-generating motors?

Department of Molecular Genetics and Cell Biology The University of Chicago Chicago, Illinois 60637 in recent years there has been intense interest in a group of proteins known as molecular chaperones (Georgopoulos and Welch, 1993). One class of molecular chaperones is the Hsp70 family, which comprises several closely related proteins of about 70 kDa (McKay et al., 1994). Members of the Hsp70 family are found in the cytosol of prokaryotes and eukaryotes and in the lumenal spaces of mitochon- dria, chloroplasts, and the endoplasmic reticulum. Hsp70 chaperones have been implicated in protein folding, the assembly and disassembly of oligcmeric complexes, pro- tein synthesis and degradation, and the translocation of polypeptides across cellular membranes. Despite this wealth of information, the mechanism of Hsp70 action is still poorly understood. Hsp70 proteins have a two-domain structure: the N-ter- minal domain binds adenine nucleotides, and the C-ter- minal domain binds short segments of extended polypep- tide (Hightower et al., 1994; McKay et al., 1994). In the presence of ADP, Hsp70 associates tightly with polypep- tide substrates. This interaction is weakened by the addi- tion of ATP. Release of polypeptides from Hsp70 is caused not by ATP hydrolysis, as was originally proposed, but rather by ATP binding (Hightower et al., 1994). It is gener- ally believed that this nucleotide-dependent cycle of poly- peptide chain binding and release can account for all of the diverse actions of Hsp70 proteins. For example, during protein folding, Hsp70 molecules might bind transiently to unstructured regions of polypeptide chain and thereby prevent the incompletely folded polypeptides from aggre- gating (Georgopoulos and Welch, 1993). Thus, Hsp70 chaperones would not serve as catalysts to speed up fold- ing, but instead would inhibit unproductive side reactions. However, as described below, recent studies of protein translocation suggest that certain functions of Hsp70 chaperones involve more than mere binding to polypep- tide substrates. It seems that Hsp70 proteins can some- times accelerate reactions that would otherwise be prohib- itively slow.

The Curious Status of the Golgi Apparatus

The available evidence suggests the following working hypotheses. (1) COPII vesicles fuse to form ERGIC clusters, which fuse in turn to form a new Golgi cisterna. (2) Each cisterna progresses through the stack and ultimately fragments into transport vesicles. (3) All resident Golgi proteins are continually recycled via retrograde vesicles. This vesicular transport drives the maturation of Golgi cisternae.Each of these hypotheses raises a new set of questions. First, what nucleates the formation of an ERGIC cluster? Is a cytosolic scaffold involved? How does the cell decide when a given ERGIC cluster has been completed, so that subsequent COPII vesicles will contribute to forming a new cluster? What determines the number of ERGIC clusters that fuse to generate a cisterna? Second, is cisternal progression an active process, or do the cisternae simply diffuse? What signal induces a cisterna to undergo terminal maturation at the TGN? Third, how do COPI vesicles incorporate all of the diverse proteins that reside in the Golgi? Are there multiple classes of COPI vesicles (Orci et al. 1997xBidirectional transport by distinct populations of COPI-coated vesicles. Orci, L, Stamnes, M, Ravazzola, M, Amherdt, M, Perrelet, A, Sollner, T.H, and Rothman, J.E. Cell. 1997; 90: 335–349Abstract | Full Text | Full Text PDF | PubMed | Scopus (296)See all ReferencesOrci et al. 1997)? In addition to COPI vesicles, other vesicles apparently retrieve proteins that cycle between the TGN and later compartments of the endomembrane system (Munro 1998xLocalization of proteins to the Golgi apparatus. Munro, S. Trends Cell Biol. 1998; 8: 11–15Abstract | Full Text PDF | PubMed | Scopus (194)See all ReferencesMunro 1998). How is this TGN recycling pathway integrated with intra-Golgi recycling?These questions, which concern the mechanics of Golgi operation, tend to mask the embarrassing fact that we still do not understand how the structure of the Golgi relates to its function. For example, why are Golgi cisternae organized into stacks in most cell types, but dispersed throughout the cytoplasm in S. cerevisiae? One can only speculate that the sites of cisternal assembly are arranged differently in S. cerevisiae than in other eukaryotes. And why do Golgi cisternae have similar diameters in all cells (Farquhar and Palade 1981xThe Golgi apparatus (complex) - (1954–1981) - from artifact to center stage. Farquhar, M.G and Palade, G.E. J. Cell Biol. 1981; 91: 77s–103sCrossref | PubMedSee all ReferencesFarquhar and Palade 1981)? A possible explanation is that if cisternae were larger, vesicular transport would be too slow to effect maturation. Vesicles bud from the cisternal rims, so the maximal rate of vesicular transport will be proportional to a cisternas diameter, whereas the surface area and volume of a cisterna are roughly proportional to the square of its diameter. Thus, the larger the cisterna, the more slowly its contents will turn over by vesicle-driven maturation.Perhaps the most fundamental mystery concerns the basic organization of the Golgi. Why is this organelle subdivided into cisternae in the first place, and why does the number of cisternae per stack range from about 3 to 30 in various cell types (Mollenhauer and Morre 1991xPerspectives on Golgi apparatus form and function. Mollenhauer, H.H and Morre, D.J. J. Electron Microsc. Tech. 1991; 17: 2–14Crossref | PubMed | Scopus (42)See all ReferencesMollenhauer and Morre 1991)? An interesting theory is that the Golgi stack serves as an iterative recycling device for bringing resident ER proteins back to the ER (Rothman and Wieland 1996xProtein sorting by transport vesicles. Rothman, J.E and Wieland, F.T. Science. 1996; 272: 227–234Crossref | PubMedSee all ReferencesRothman and Wieland 1996). However, resident ER proteins rarely travel beyond the first cisterna or two (Pelham 1995xSorting and retrieval between the endoplasmic reticulum and Golgi apparatus. Pelham, H. Curr. Opin. Cell Biol. 1995; 7: 530–535Crossref | PubMed | Scopus (130)See all ReferencesPelham 1995), so it seems unlikely that cells would need extended Golgi stacks solely to recycle ER proteins. An alternative idea comes from recognizing that the Golgi is a carbohydrate factory, with the cisternal stack being analogous to an assembly line (39xThe Golgi complex (in vitro veritas?) . Mellman, I and Simons, K. Cell. 1992; 68: 829–840Abstract | Full Text PDF | PubMed | Scopus (306)See all References, 28xThe molecular and cell biology of glycosyltransferases. Kleene, R and Berger, E.G. Biochim. Biophys. Acta. 1993; 1154: 283–325Crossref | PubMed | Scopus (173)See all References). Secretory products need to spend enough time on the assembly line for all of the carbohydrate processing steps to be completed. By forcing a cisterna to progress through the entire stack, a cell ensures that the secretory cargo remains in the Golgi long enough to be fully processed (i.e, the stack serves as a “delay timer”). In support of this notion, the number of cisternae in a stack seems to correlate with the complexity of the secretory products being produced by the cell (Becker and Melkonian 1996xThe secretory pathway of protists (spatial and functional organization and evolution) . Becker, B and Melkonian, M. Microbiol. Rev. 1996; 60: 697–721PubMedSee all ReferencesBecker and Melkonian 1996).The new dynamic view of the Golgi may also be applicable to endosomes. Indeed, maturation models have been discussed extensively in the endosome field (24xEndosomes. Helenius, A, Mellman, I, Wall, D, and Hubbard, A. Trends Biochem. Sci. 1983; 8: 245–250Abstract | Full Text PDF | Scopus (248)See all References, 20xMembrane transport in the endocytic pathway. Gruenberg, J and Maxfield, F.R. Curr. Opin. Cell Biol. 1995; 7: 552–563Crossref | PubMed | Scopus (508)See all References). One interpretation of the existing data is that endocytic vesicles fuse homotypically to form early endosomes, which mature into late endosomes and eventually fuse with lysosomes. Hence, many compartments of the endomembrane system seem to be transitory structures that can only be understood in the context of other interacting compartments. So how then do we define the Golgi? Where does it begin and end? Seen in this light, the Golgi acquires a curious status: it is more than just an outgrowth of the ER, yet it is not truly an independent organelle.Is the cisternal maturation model the last word on Golgi organization? Almost certainly not. There is still no consensus about any feature of this model. For example, some investigators propose that COPI vesicles mediate anterograde as well as retrograde transport (Orci et al. 1997xBidirectional transport by distinct populations of COPI-coated vesicles. Orci, L, Stamnes, M, Ravazzola, M, Amherdt, M, Perrelet, A, Sollner, T.H, and Rothman, J.E. Cell. 1997; 90: 335–349Abstract | Full Text | Full Text PDF | PubMed | Scopus (296)See all ReferencesOrci et al. 1997). Even more complicated models emerge from the debate about whether tubular connections between cisternae play a role in transport through the stack (39xThe Golgi complex (in vitro veritas?) . Mellman, I and Simons, K. Cell. 1992; 68: 829–840Abstract | Full Text PDF | PubMed | Scopus (306)See all References, 40xVariations on the intracellular transport theme (maturing cisternae and trafficking tubules) . Mironov, A.A, Weidman, P, and Luini, A. J. Cell Biol. 1997; 138: 481–484Crossref | PubMed | Scopus (117)See all References). In his 1975 monograph, Whaley wrote, “No cellular organelle has been the subject of as many, as long-lasting, or as diverse polemics as the Golgi apparatus.” We are happy to report that this statement is just as true in 1998.‡To whom correspondence should be addressed (e-mail: malhotra@biomail.ucsd.edu).

De novo formation of transitional ER sites and Golgi structures in Pichia pastoris.

Transitional ER (tER) sites are ER subdomains that are functionally, biochemically and morphologically distinct from the surrounding rough ER. Here we have used confocal video microscopy to study the dynamics of tER sites and Golgi structures in the budding yeast Pichia pastoris. The biogenesis of tER sites is tightly linked to the biogenesis of Golgi, and both compartments can apparently form de novo. tER sites often fuse with one another, but they maintain a consistent average size through shrinkage after fusion and growth after de novo formation. Golgi dynamics are similar, although late Golgi elements often move away from tER sites towards regions of polarized growth. Our results can be explained by assuming that tER sites give rise to Golgi cisternae that continually mature.

Membrane traffic within the Golgi apparatus.

Newly synthesized secretory cargo molecules pass through the Golgi apparatus while resident Golgi proteins remain in the organelle. However, the pathways of membrane traffic within the Golgi are still uncertain. Most of the available data can be accommodated by the cisternal maturation model, which postulates that Golgi cisternae form de novo, carry secretory cargoes forward and ultimately disappear. The entry face of the Golgi receives material that has been exported from transitional endoplasmic reticulum sites, and the exit face of the Golgi is intimately connected with endocytic compartments. These conserved features are enhanced by cell-type-specific elaborations such as tubular connections between mammalian Golgi cisternae. Key mechanistic questions remain about the formation and maturation of Golgi cisternae, the recycling of resident Golgi proteins, the origins of Golgi compartmental identity, the establishment of Golgi architecture, and the roles of Golgi structural elements in membrane traffic.

Fatty acyl-coenzyme a is required for budding of transport vesicles from Golgi cisternae

We describe a new role for fatty acylation. Conditions were established under which vesicular transport from the cis to the medial Golgi compartment in vitro depends strongly upon the addition of a fatty acyl-coenzyme A, e.g., palmitoyl-CoA. Using an inhibitor of long-chain acyl-CoA synthetase, we demonstrate that the fatty acid has to be activated by CoA to stimulate transport. A nonhydrolyzable analog of palmitoyl-CoA competitively inhibits transport. Electron microscopy and biochemical studies show that fatty acyl-CoA is required for budding of (non-clathrin-) coated transport vesicles from Golgi cisternae and that budding is inhibited by the nonhydrolyzable analog.

Protein sorting in mitochondria

Most polypeptides that are imported into the mitochondrial matrix use a common translocation machinery. By contrast, proteins of the other mitochondrial compartments are imported by a variety of different mechanisms. Some of these proteins completely bypass the common translocation machinery, others use only the outer membrane components of this machinery, and still others use components of this machinery from both the outer and inner membranes. Import to the intermembrane space compartment provides examples of all three possibilities.

A mitochondrial homolog of bacterial GrpE interacts with mitochondrial hsp70 and is essential for viability.

Mitochondrial hsp70 (mhsp70) is located in the matrix and an essential component of the mitochondrial protein import system. To study the function of mhsp70 and to identify possible partner proteins we constructed a yeast strain in which all mhsp70 molecules carry a C‐terminal hexa‐histidine tag. The tagged mhsp70 appears to be functional in vivo. When an ATP depleted mitochondrial extract was incubated with a nickel‐derivatized affinity resin, the resin bound not only mhsp70, but also a 23 kDa protein. This protein was dissociated from mhsp70 by ATP. ADP and GTP were much less effective in promoting dissociation whereas CTP and TTP were inactive. We cloned the gene encoding the 23 kDa protein. This gene, termed GRPE, encodes a 228 residue protein, whose sequence closely resembles that of the bacterial GrpE protein. Microsequencing the purified 23 kDa protein established it as the product of the yeast GRPE gene. Yeast GrpEp is made as a precursor that is cleaved upon import into isolated mitochondria. GrpEp is essential for viability. We suggest that this protein interacts with mhsp70 in a manner analogous to that of GrpE with DnaK of E.coli.