Ypt/rab gtpases: regulators of protein trafficking.

Ypt/Rab guanosine triphosphatases (GTPases) have emerged in the last decade as key regulators of protein transport in all eukaryotic cells. They seem to be involved in all aspects of vesicle trafficking: vesicle formation, motility, and docking, and membrane remodeling and fusion. The functions of Ypt/Rabs are themselves controlled by upstream regulators that stimulate both their nucleotide cycling and their cycling between membranes. Ypt/Rabs transmit signals to downstream effectors in a guanosine triphosphate (GTP)-dependent manner. The identity of upstream regulators and downstream effectors is known for a number of Ypt/Rabs, and models for their mechanisms of action are emerging. In at least two cases, Ypt/Rab upstream regulators and downstream effectors are found together in a single complex. In agreement with the idea that Ypt/Rabs function in all aspects of vesicular transport, their diverse effectors have recently been shown to function in all identified aspects of vesicle transport. Activators and effectors for individual Ypt/Rabs share no similarity, but are conserved between yeast and mammalian cells. Finally, cross talk demonstrated among the various Ypt/Rabs, and between Ypt/Rabs and other signaling factors, suggests possible coordination among secretory steps, as well as between protein transport and other cellular processes.

Ypt and Rab GTPases: insight into functions through novel interactions.

Ypt/Rab GTPases are key regulators of vesicular transport in eukaryotic cells. During the past two years, a number of new Ypt/Rab-interacting proteins have been identified and shown to serve as either upstream regulators or downstream effectors. Proteins that interact with these regulators and effectors of Ypt/Rabs have also been identified, and together they provide new insights into Ypt/Rab mechanisms of action. The picture that emerges from these studies suggests that Ypt/Rabs function in multiple and diverse aspects of vesicular transport. In addition, not only are Ypt/Rabs highly conserved, but their functions and interactions are as well. Interestingly, crosstalk among Ypt/Rabs and between Ypt/Rabs and other signaling factors, suggest the possibility of coordination of the individual vesicular transport steps and of the protein transport machinery with other cellular processes.

The TRAPP complex is a nucleotide exchanger for Ypt1 and Ypt31/32.

In yeast, the Ypt1 GTPase is required for ER-to-cis-Golgi and cis-to-medial-Golgi protein transport, while Ypt31/32 are a functional pair of GTPases essential for exit from the trans-Golgi. We have previously identified a Ypt1 guanine nucleotide exchange factor (GEF) activity and characterized it as a large membrane-associated protein complex that localizes to the Golgi and can be extracted from the membrane by salt, but not by detergent. TRAPP is a large protein complex that is required for ER-to-Golgi transport and that has properties similar to those of Ypt1 GEF. Here we show that TRAPP has Ypt1 GEF activity. GST-tagged Bet3p or Bet5p, two of the TRAPP subunits, were expressed in yeast cells and were precipitated by glutathione-agarose (GA) beads. The resulting precipitates can stimulate both GDP release and GTP uptake by Ypt1p. The majority of the Ypt1 GEF activity associated with the GST-Bet3p precipitate has an apparent molecular weight of > 670 kDa, indicating that the GEF activity resides in the TRAPP complex. Surprisingly, TRAPP can also stimulate nucleotide exchange on the Ypt31/32 GTPases, but not on Sec4p, a Ypt-family GTPase required for the last step of the exocytic pathway. Like the previously characterized Ypt1 GEF, the TRAPP Ypt1-GEF activity can be inhibited by the nucleotide-free Ypt1-D124N mutant protein. This mutant protein also inhibits the Ypt32 GEF activity of TRAPP. Coprecipitation and overexpression studies suggest that TRAPP can act as a GEF for Ypt1 and Ypt31/32 in vivo. These data suggest the exciting possibility that a GEF complex common to Ypt1 and Ypt31/32 might coordinate the function of these GTPases in entry into and exit from the Golgi.

Genetic interactions in yeast between Ypt GTPases and Arf guanine nucleotide exchangers.

Two families of GTPases, Arfs and Ypt/rabs, are key regulators of vesicular transport. While Arf proteins are implicated in vesicle budding from the donor compartment, Ypt/rab proteins are involved in the targeting of vesicles to the acceptor compartment. Recently, we have shown a role for Ypt31/32p in exit from the yeast trans-Golgi, suggesting a possible function for Ypt/rab proteins in vesicle budding as well. Here we report the identification of a new member of the Sec7-domain family, SYT1, as a high-copy suppressor of a ypt31/32 mutation. Several proteins that belong to the Sec7-domain family, including the yeast Gea1p, have recently been shown to stimulate nucleotide exchange by Arf GTPases. Nucleotide exchange by Arf GTPases, the switch from the GDP- to the GTP-bound form, is thought to be crucial for their function. Sec7p itself has an important role in the yeast secretory pathway. However, its mechanism of action is not yet understood. We show that all members of the Sec7-domain family exhibit distinct genetic interactions with the YPT genes. Biochemical assays demonstrate that, although the homology between the members of the Sec7-domain family is relatively low (20-35%) and limited to a small domain, they all can act as guanine nucleotide exchange factors (GEFs) for Arf proteins, but not for Ypt GTPases. The Sec7-domain of Sec7p is sufficient for this activity. Interestingly, the Sec7 domain activity is inhibited by brefeldin A (BFA), a fungal metabolite that inhibits some of the Arf-GEFs, indicating that this domain is a target for BFA. These results demonstrate that the ability to act as Arf-GEFs is a general property of all Sec7-domain proteins in yeast. The genetic interactions observed between Arf GEFs and Ypt GTPases suggest the existence of a Ypt-Arf GTPase cascade in the secretory pathway.

Identification of regulators for Ypt1 GTPase nucleotide cycling.

Small GTPases of the Ypt/Rab family are involved in the regulation of vesicular transport. Cycling between the GDP- and GTP-bound forms and the accessory proteins that regulate this cycling are thought to be crucial for Ypt/Rab function. Guanine nucleotide exchange factors (GEFs) stimulate both GDP loss and GTP uptake, and GTPase-activating proteins (GAPs) stimulate GTP hydrolysis. Little is known about GEFs and GAPs for Ypt/Rab proteins. In this article we report the identification and initial characterization of two factors that regulate nucleotide cycling by Ypt1p, which is essential for the first two steps of the yeast secretory pathway. The Ypt1p-GEF stimulates GDP release and GTP uptake at least 10-fold and is specific for Ypt1p. Partially purified Ypt1p-GEF can rescue the inhibition caused by the dominant-negative Ypt1p-D124N mutant of in vitro endoplasmic reticulum-to-Golgi transport. This mutant probably blocks transport by inhibiting the GEF, suggesting that we have identified the physiological GEF for Ypt1p. The Ypt1p-GAP stimulates GTP hydrolysis by Ypt1p up to 54-fold, has a higher affinity for the GTP-bound form of Ypt1p than for the GDP-bound form, and is specific to a subgroup of exocytic Ypt proteins. The Ypt1p-GAP activity is not affected by deletion of two genes that encode known Ypt GAPs, GYP7 and GYP1, nor is it influenced by mutations in SEC18, SEC17, or SEC22, genes whose products are involved in vesicle fusion. The GEF and GAP activities for Ypt1p localize to particulate cellular fractions. However, contrary to the predictions of current models, the GEF activity localizes to the fraction that functions as the acceptor in an endoplasmic reticulum-to-Golgi transport assay, whereas the GAP activity cofractionates with markers for the donor. On the basis of our current and previous results, we propose a new model for the role of Ypt/Rab nucleotide cycling and the factors that regulate this process.

GTP hydrolysis is not important for Ypt1 GTPase function in vesicular transport.

GTPases of the Ypt/Rab family play a key role in the regulation of vesicular transport. Their ability to cycle between the GTP- and the GDP-bound forms is thought to be crucial for their function. Conversion from the GTP- to the GDP-bound form is achieved by a weak endogenous GTPase activity, which can be stimulated by a GTPase-activating protein (GAP). Current models suggest that GTP hydrolysis and GAP activity are essential for vesicle fusion with the acceptor compartment or for timing membrane fusion. To test this idea, we inactivated the GTPase activity of Ypt1p by using the Q67L mutation, which targets a conserved residue that helps catalyze GTP hydrolysis in Ras. We demonstrate that the mutant Ypt1-Q67L protein is severely impaired in its ability to hydrolyze GTP both in the absence and in the presence of GAP and consequently is restricted mostly to the GTP-bound form. Surprisingly, a strain with ypt1-Q67L as the only YPT1 gene in the cell has no observable growth phenotypes at temperatures ranging from 14 to 37 degrees C. In addition, these mutant cells exhibit normal rates of secretion and normal membrane morphology as determined by electron microscopy. Furthermore, the ypt1-Q67L allele does not exhibit dominant phenotypes in cell growth and secretion when overexpressed. Together, these results lead us to suggest that, contrary to current models for Ypt/Rab function, GTP hydrolysis is not essential either for Ypt1p-mediated vesicular transport or as a timer to turn off Ypt1p-mediated membrane fusion but only for recycling of Ypt1p between compartments. Finally, the ypt1-Q67L allele, like the wild type, is inhibited by dominant nucleotide-free YPT1 mutations. Such mutations are thought to exert their dominant phenotype by sequestration of the guanine nucleotide exchange factor (GNEF). These results suggest that the function of Ypt1p in vesicular transport requires not only the GTP-bound form of the protein but also the interaction of Ypt1p with its GNEF.

Two new Ypt GTPases are required for exit from the yeast trans-Golgi compartment.

Small GTPases of the Ypt/rab family are involved in the regulation of vesicular transport. These GTPases apparently function during the targeting of vesicles to the acceptor compartment. Two members of the Ypt/rab family, Ypt1p and Sec4p, have been shown to regulate early and late steps of the yeast exocytic pathway, respectively. Here we tested the role of two newly identified GTPases, Ypt31p and Ypt32p. These two proteins share 81% identity and 90% similarity, and belong to the same protein subfamily as Ypt1p and Sec4p. Yeast cells can tolerate deletion of either the YPT31 or the YPT32 gene, but not both. These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions. Cells deleted for the YPT31 gene and carrying a conditional ypt32 mutation exhibit protein transport defects in the late exocytic pathway, but not in vacuolar protein sorting. The ypt31/ 32 mutant secretory defect is clearly downstream from that displayed by a ypt1 mutant and is similar to that of sec4 mutant cells. However, electron microscopy revealed that while sec4 mutant cells accumulate secretory vesicles, ypt31/32 mutant cells accumulate aberrant Golgi structures. The ypt31/32 phenotype is epistatic to that of a sec1 mutant, which accumulates secretory vesicles. Together, these results indicate that the Ypt31/32p GTPases are required for a step that occurs in the trans-Golgi compartment, between the reactions regulated by Ypt1p and Sec4p. This step might involve budding of vesicles from the trans-Golgi. Alternatively, Ypt31/32p might promote secretion indirectly, by allowing fusion of recycling vesicles with the trans-Golgi compartment.

The Ypt1 GTPase is essential for the first two steps of the yeast secretory pathway.

Small GTPases of the rab family are involved in the regulation of vesicular transport. The restricted distribution of each of these proteins in mammalian cells has led to the suggestion that different rab proteins act at different steps of transport (Pryer, N. K., L. J. Wuestehube, and R. Sheckman. 1992. Annu Rev. Biochem. 61:471-516; Zerial, M., and H. Stenmark. 1993. Curr. Opin. Cell Biol. 5:613-620). However, in this report we show that the Ypt1-GTPase, a member of the rab family, is essential for more than one step of the yeast secretory pathway. We determined the secretory defect conferred by a novel ypt1 mutation by comparing the processing of several transported glycoproteins in wild-type and mutant cells. The ypt1-A136D mutant has a change in an amino acid that is conserved among rab GTPases. This mutation leads to a rapid and tight secretory block upon a shift to the restrictive temperature, and allows for the identification of the specific steps in the secretory pathway that directly require Ypt1 protein (Ypt1p). The ypt1-A136D mutant exhibits tight blocks in two secretory steps, ER to cis-Golgi and cis- to medial-Golgi, but later steps are unaffected. Thus, it is unlikely that Ypt1p functions as the sole determinant of fusion specificity. Our results are more consistent with a role for Ypt1/rab proteins in determining the directionality or fidelity of protein sorting.

Requirement of nucleotide exchange factor for Ypt1 GTPase mediated protein transport.

Small GTPases of the rab family are involved in the regulation of vesicular transport. It is believed that cycling between the GTP- and GDP-bound forms, and accessory factors regulating this cycling are crucial for rab function. However, an essential role for rab nucleotide exchange factors has not yet been demonstrated. In this report we show the requirement of nucleotide exchange factor activity for Ypt1 GTPase mediated protein transport. The Ypt1 protein, a member of the rab family, plays a role in targeting vesicles to the acceptor compartment and is essential for the first two steps of the yeast secretory pathway. We use two YPT1 dominant mutations that contain alterations in a highly conserved GTP-binding domain, N121I and D124N. YPT1-D124N is a novel mutation that encodes a protein with nucleotide specificity modified from guanine to xanthine. This provides a tool for the study of an individual rab GTPase in crude extracts: a xanthosine triphosphate (XTP)-dependent conditional dominant mutation. Both mutations confer growth inhibition and a block in protein secretion when expressed in vivo. The purified mutant proteins do not bind either GDP or GTP. Moreover, they completely inhibit the ability of the exchange factor to stimulate nucleotide exchange for wild type Ypt1 protein, and are potent inhibitors of ER to Golgi transport in vitro at the vesicle targeting step. The inhibitory effects of the Ypt1-D124N mutant protein on both nucleotide exchange activity and protein transport in vitro can be relieved by XTP, indicating that it is the nucleotide-free form of the mutant protein that is inhibitory. These results suggest that the dominant mutant proteins inhibit protein transport by sequestering the exchange factor from the wild type Ypt1 protein, and that this factor has an essential role in vesicular transport.

Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy.

The membrane compartments responsible for Golgi functions in wild-type Saccharomyces cerevisiae were identified and characterized by immunoelectron microscopy. Using improved fixation methods, Golgi compartments were identified by labeling with antibodies specific for alpha 1-6 mannose linkages, the Sec7 protein, or the Ypt1 protein. The compartments labeled by each of these antibodies appear as disk-like structures that are apparently surrounded by small vesicles. Yeast Golgi typically are seen as single, isolated cisternae, generally not arranged into parallel stacks. The location of the Golgi structures was monitored by immunoelectron microscopy through the yeast cell cycle. Several Golgi compartments, apparently randomly distributed, were always observed in mother cells. During the initiation of new daughter cells, additional Golgi structures cluster just below the site of bud emergence. These Golgi enter daughter cells at an early stage, raising the possibility that much of the bud's growth might be due to secretory vesicles formed as well as consumed entirely within the daughter. During cytokinesis, the Golgi compartments are concentrated near the site of cell wall synthesis. Clustering of Golgi both at the site of bud formation and at the cell septum suggests that these organelles might be directed toward sites of rapid cell surface growth.

Mediation of the attachment or fusion step in vesicular transport by the GTP-binding Ypt1 protein.

The function of the guanosine triphosphate (GTP)-binding protein Ypt1 in regulating vesicular traffic was studied in a cell-free system that reconstitutes transport from the endoplasmic reticulum to the Golgi. Blocking the Ypt1 protein activity resulted in accumulation of vesicles that act as an intermediate passing between the two compartments. The Ypt1 protein was found on the outer side of these vesicles. The transport process is completed by fusion of these vesicles with the acceptor compartment, and Ypt1 protein activity was needed for this step. Thus, a specific GTP-binding protein is required for either attachment or fusion (or both) of secretory vesicles with the acceptor compartment during protein secretion.

GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction.

The 21-kDa GTP-binding Ypt1 protein (Ypt1p) is required for protein transport from the endoplasmic reticulum to the Golgi complex in yeast extracts. Ypt1 antibodies block transport; this inhibition is alleviated by competition with excess purified Ypt1p produced in bacteria. Furthermore, extracts of cells carrying the mutation ypt1-1 are defective in transport, but transport is restored if a cytosolic fraction from wild-type cells is provided. The in vitro transport reaction also requires physiological levels of Ca2+. However, Ypt1p functions independently of Ca2+. First, buffering the free Ca2+ at concentrations ranging from 1 nM to 10 microM does not relieve inhibition by Ypt1 antibodies. Second, consumption of a Ca2+-requiring intermediate that accumulates in Ca2+-deficient incubations is not inhibited by anti-Ypt1 antibodies, although completion of transport requires ATP and an N-ethylmaleimide-sensitive factor. Thus, Ypt1p and Ca2+ are required at distinct steps.

The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery.

A yeast GTP-binding protein, the YPT1 gene product, has been found to function early in the secretion pathway. The ypt1-1 mutation causes a phenotype reminiscent of early secretion-defective mutants, including accumulation of membranes and vesicles as well as a partial defect in secretion and incomplete glycosylation of invertase. Immunofluorescence localization studies using affinity-purified antibody directed against the YPT1 protein showed punctate staining of the cytoplasm of growing yeast cells and very intense staining of small buds, where membrane growth and secretion are most active. The punctate cytoplasmic staining is changed in a mutant (sec7) under conditions that cause aberrant Golgi structures to accumulate. The pattern of immunofluorescence obtained when mouse cells were stained with the antibody coincided closely with the pattern observed with wheat germ agglutinin, suggesting that a mammalian counterpart of the yeast YPT1 protein is located in the Golgi apparatus. These results are interpreted as suggesting that GTP-binding proteins may act to direct intracellular vesicle traffic.

The ras-like yeast YPT1 gene is itself essential for growth, sporulation, and starvation response.

The Saccharomyces cerevisiae gene YPT1 encodes a protein that exhibits significant homology to the mammalian ras proteins. Using gene disruption techniques, we have shown that the intact YPT1 gene is required for spore viability. Lethality caused by loss of YPT1 function, unlike that caused by loss of the yeast ras homologs RAS1 and RAS2 function, is not suppressed by the bcy1 mutation, suggesting that YPT1 does not act through the adenylate cyclase regulatory system. A cold-sensitive allele, ypt1-1, was constructed. At the nonpermissive temperature, mutants died, exhibiting aberrant nuclear morphology, as well as abnormal distribution of actin and tubulin. The mutant cells died without exhibiting classical cell-cycle-specific arrest; nevertheless, examination of cellular DNA content suggests that the YPT1 function is required, particularly after S phase. Cells carrying the ypt1-1 mutation died upon nitrogen starvation even at a temperature permissive for growth; diploid cells homozygous for ypt1-1 did not sporulate. The YPT1 gene is thus involved in nutritional regulation of the cell cycle as well as in normal progression through the mitotic cell cycle.

Characterization of a protein, released by the T47D cell line, immunologically related to the major envelope protein of mouse mammary tumor virus.

The T47D human mammary adenocarcinoma cell line in vitro releases viral particles as well as soluble proteins. Both fractions were shown to contain antigens that immunologically crossreact with the major glycoprotein (gp52) of mouse mammary tumor virus. The crossreacting antigens are located on polypeptides with apparent molecular weights of about 68,000 and 60,000. The larger one is present in viral particles whereas both are found in the soluble fraction. Both proteins are glycosylated. The human tissue culture proteins differ from gp52 not only in molecular weight but also in charge heterogenity and in polypeptide profiles obtained after partial proteolysis. The results suggest that there is a restricted similarity between MMTV gp52 and the immunologically related T47D proteins.