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

Jedd G, Mulholland J, Segev N - J. Cell Biol. (1997)

Bottom Line: These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological and Physiological Sciences, The University of Chicago, Illinois 60637, USA.

ABSTRACT
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.

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ypt31-Δ/ypt32A141D mutant cells accumulate aberrant Golgi-like  structures at the nonpermissive temperature. Wild-type  and three strains mutated at  the analogous conserved residue in YPT1, YPT32, and  SEC4 were analyzed by electron microscopy. Electron  micrographs of representative cells are shown for the  following strains: (A) wildtype (NSY128), (B) ypt1A136D (NSY222), (C) ypt31Δ/ypt32-A141D (NSY348),  and (D) sec4-G147D (PNY  404). Cells were grown at  26°C, shifted to 37°C for 2 h,  and then processed for thin  section electron microscopy.  Arrows point to membranous structures unique to  each strain. Arrowheads indicate ER. G, wild-type Golgi  cisterna (shown in detail in  Fig. 9 A, second panel from  left); V, vacuole; n, nucleus.  (E) Quantification of the distinct membranous structures  that accumulate in the different mutant strains: small vesicles (50–80 nm), Golgi (cisternae or Berkeley bodies),  and large vesicles (100–150  nm). Bars represent the  mean number of structures in  30 cell sections. Data is normalized to density per cubic  micrometer for vesicle populations and density per 10  μm2 for cisternae and Berkeley bodies. Error bars represent one standard deviation  (see Materials and Methods  for details of the quantification procedure). The relatively high standard deviations are probably due to the  aggregation of the aberrant  membranes to one side of the  cell, resulting in cell sections  that are either rich in or devoid of the corresponding  membranous structure. Bar,  1 μm.
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Figure 7: ypt31-Δ/ypt32A141D mutant cells accumulate aberrant Golgi-like structures at the nonpermissive temperature. Wild-type and three strains mutated at the analogous conserved residue in YPT1, YPT32, and SEC4 were analyzed by electron microscopy. Electron micrographs of representative cells are shown for the following strains: (A) wildtype (NSY128), (B) ypt1A136D (NSY222), (C) ypt31Δ/ypt32-A141D (NSY348), and (D) sec4-G147D (PNY 404). Cells were grown at 26°C, shifted to 37°C for 2 h, and then processed for thin section electron microscopy. Arrows point to membranous structures unique to each strain. Arrowheads indicate ER. G, wild-type Golgi cisterna (shown in detail in Fig. 9 A, second panel from left); V, vacuole; n, nucleus. (E) Quantification of the distinct membranous structures that accumulate in the different mutant strains: small vesicles (50–80 nm), Golgi (cisternae or Berkeley bodies), and large vesicles (100–150 nm). Bars represent the mean number of structures in 30 cell sections. Data is normalized to density per cubic micrometer for vesicle populations and density per 10 μm2 for cisternae and Berkeley bodies. Error bars represent one standard deviation (see Materials and Methods for details of the quantification procedure). The relatively high standard deviations are probably due to the aggregation of the aberrant membranes to one side of the cell, resulting in cell sections that are either rich in or devoid of the corresponding membranous structure. Bar, 1 μm.

Mentions: The fact that the secretory defect of ypt31/32 mutant cells is similar to that of sec4 cells could indicate that Ypt31/32 and Sec4 GTPases function in the same process, i.e., fusion of secretory vesicles with the plasma membrane. Alternatively, they might have roles in two different secretory steps or substeps, e.g., Ypt31/32 might function in vesicle budding or fusion at the trans-Golgi compartment, and Sec4 in fusion of trans-Golgi–derived vesicles with the plasma membrane. In general, sec mutants have been shown to accumulate aberrant membranes of secretory compartments that precede the step in which they first function (Novick et al., 1981; Kaiser and Schekman, 1990). Thus, sec4 mutant cells have been shown to accumulate secretory vesicles (Novick et al., 1980; Walworth et al., 1989). To determine whether ypt31/32 mutant cells accumulate such vesicles or whether they accumulate other membranous structures, cells were shifted from the permissive temperature (26°C) to the nonpermissive temperature (37°C) for 2 h and examined by electron microscopy. We quantified the accumulation of various aberrant membrane structures: small vesicles (50–80 nm), aberrant Golgi (cisternae and Berkeley bodies), and large vesicles (100–150 nm) (Fig. 7 E). We compared the effect of the ypt1-A136D mutation and the analogous mutations in YPT31/32 and SEC4 on the type of membrane accumulated. Unlike sec4-G147D mutant cells, which accumulate post-Golgi vesicles almost exclusively (Fig. 7, D and E), ypt31Δ/32-A141D mutant cells exhibited an abundance of aberrant Golgi-like structures (Fig. 7, C and E) and some ER (Fig. 7 C, arrowhead). This phenotype is also distinct from that observed for ypt1A136D mutant cells, which accumulate ER membranes and small secretory vesicles (Fig. 7 B), but not Golgi.


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

Jedd G, Mulholland J, Segev N - J. Cell Biol. (1997)

ypt31-Δ/ypt32A141D mutant cells accumulate aberrant Golgi-like  structures at the nonpermissive temperature. Wild-type  and three strains mutated at  the analogous conserved residue in YPT1, YPT32, and  SEC4 were analyzed by electron microscopy. Electron  micrographs of representative cells are shown for the  following strains: (A) wildtype (NSY128), (B) ypt1A136D (NSY222), (C) ypt31Δ/ypt32-A141D (NSY348),  and (D) sec4-G147D (PNY  404). Cells were grown at  26°C, shifted to 37°C for 2 h,  and then processed for thin  section electron microscopy.  Arrows point to membranous structures unique to  each strain. Arrowheads indicate ER. G, wild-type Golgi  cisterna (shown in detail in  Fig. 9 A, second panel from  left); V, vacuole; n, nucleus.  (E) Quantification of the distinct membranous structures  that accumulate in the different mutant strains: small vesicles (50–80 nm), Golgi (cisternae or Berkeley bodies),  and large vesicles (100–150  nm). Bars represent the  mean number of structures in  30 cell sections. Data is normalized to density per cubic  micrometer for vesicle populations and density per 10  μm2 for cisternae and Berkeley bodies. Error bars represent one standard deviation  (see Materials and Methods  for details of the quantification procedure). The relatively high standard deviations are probably due to the  aggregation of the aberrant  membranes to one side of the  cell, resulting in cell sections  that are either rich in or devoid of the corresponding  membranous structure. Bar,  1 μm.
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Figure 7: ypt31-Δ/ypt32A141D mutant cells accumulate aberrant Golgi-like structures at the nonpermissive temperature. Wild-type and three strains mutated at the analogous conserved residue in YPT1, YPT32, and SEC4 were analyzed by electron microscopy. Electron micrographs of representative cells are shown for the following strains: (A) wildtype (NSY128), (B) ypt1A136D (NSY222), (C) ypt31Δ/ypt32-A141D (NSY348), and (D) sec4-G147D (PNY 404). Cells were grown at 26°C, shifted to 37°C for 2 h, and then processed for thin section electron microscopy. Arrows point to membranous structures unique to each strain. Arrowheads indicate ER. G, wild-type Golgi cisterna (shown in detail in Fig. 9 A, second panel from left); V, vacuole; n, nucleus. (E) Quantification of the distinct membranous structures that accumulate in the different mutant strains: small vesicles (50–80 nm), Golgi (cisternae or Berkeley bodies), and large vesicles (100–150 nm). Bars represent the mean number of structures in 30 cell sections. Data is normalized to density per cubic micrometer for vesicle populations and density per 10 μm2 for cisternae and Berkeley bodies. Error bars represent one standard deviation (see Materials and Methods for details of the quantification procedure). The relatively high standard deviations are probably due to the aggregation of the aberrant membranes to one side of the cell, resulting in cell sections that are either rich in or devoid of the corresponding membranous structure. Bar, 1 μm.
Mentions: The fact that the secretory defect of ypt31/32 mutant cells is similar to that of sec4 cells could indicate that Ypt31/32 and Sec4 GTPases function in the same process, i.e., fusion of secretory vesicles with the plasma membrane. Alternatively, they might have roles in two different secretory steps or substeps, e.g., Ypt31/32 might function in vesicle budding or fusion at the trans-Golgi compartment, and Sec4 in fusion of trans-Golgi–derived vesicles with the plasma membrane. In general, sec mutants have been shown to accumulate aberrant membranes of secretory compartments that precede the step in which they first function (Novick et al., 1981; Kaiser and Schekman, 1990). Thus, sec4 mutant cells have been shown to accumulate secretory vesicles (Novick et al., 1980; Walworth et al., 1989). To determine whether ypt31/32 mutant cells accumulate such vesicles or whether they accumulate other membranous structures, cells were shifted from the permissive temperature (26°C) to the nonpermissive temperature (37°C) for 2 h and examined by electron microscopy. We quantified the accumulation of various aberrant membrane structures: small vesicles (50–80 nm), aberrant Golgi (cisternae and Berkeley bodies), and large vesicles (100–150 nm) (Fig. 7 E). We compared the effect of the ypt1-A136D mutation and the analogous mutations in YPT31/32 and SEC4 on the type of membrane accumulated. Unlike sec4-G147D mutant cells, which accumulate post-Golgi vesicles almost exclusively (Fig. 7, D and E), ypt31Δ/32-A141D mutant cells exhibited an abundance of aberrant Golgi-like structures (Fig. 7, C and E) and some ER (Fig. 7 C, arrowhead). This phenotype is also distinct from that observed for ypt1A136D mutant cells, which accumulate ER membranes and small secretory vesicles (Fig. 7 B), but not Golgi.

Bottom Line: These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological and Physiological Sciences, The University of Chicago, Illinois 60637, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus