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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 Golgi membranes at the  permissive temperature and  Berkeley bodies at the nonpermissive temperature.  Electron microscopy of (A)  wild-type (NSY128), (B)  ypt31-Δ/ypt32-A141D mutant strain (NSY348) at 26°C,  and (C) ypt31-Δ/ypt32A141D mutant strain  (NSY348) at 37°C. Arrows  indicate a single Golgi cisterna in a wild-type cell and  regions of Golgi accumulation in mutant cells. v, vacuole; n, nucleus. (D) Quantification of cisternal profiles  and Berkeley bodies in wildtype cells at 26°C and ypt31Δ/ypt32-A141D mutant  (NSY348) cells at 26° and  37°C. The percentage of the  total structures counted that  are cisternal is indicated by  the gray bar, and the percentage of Berkeley bodies is  represented by the black bar.  At the permissive temperature, the mutant shows a fivefold increase in the frequency of Golgi profiles, the  majority of which (70% of  total) are cisternal. After 2 h at 37°C, the number of Golgi profiles has doubled and the population is dominated by Berkeley bodies  (60% of total). (E) Immunofluorescence microscopy using anti-Ypt1p antibodies (Segev et al., 1988). (1) wild-type cells (NSY128)  grown at 26°C; (2) the ypt31-Δ/ypt32-A141D mutant (NSY348) grown at 26°C; or (3) shifted to 37°C, for 90 min; (4) the same cells  shown in 3 photographed with Nomarski optics to show the contours of the cells. Bars: (A–C) 1 μm; (E) 10 μm.
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Figure 8: ypt31-Δ/ypt32A141D mutant cells accumulate Golgi membranes at the permissive temperature and Berkeley bodies at the nonpermissive temperature. Electron microscopy of (A) wild-type (NSY128), (B) ypt31-Δ/ypt32-A141D mutant strain (NSY348) at 26°C, and (C) ypt31-Δ/ypt32A141D mutant strain (NSY348) at 37°C. Arrows indicate a single Golgi cisterna in a wild-type cell and regions of Golgi accumulation in mutant cells. v, vacuole; n, nucleus. (D) Quantification of cisternal profiles and Berkeley bodies in wildtype cells at 26°C and ypt31Δ/ypt32-A141D mutant (NSY348) cells at 26° and 37°C. The percentage of the total structures counted that are cisternal is indicated by the gray bar, and the percentage of Berkeley bodies is represented by the black bar. At the permissive temperature, the mutant shows a fivefold increase in the frequency of Golgi profiles, the majority of which (70% of total) are cisternal. After 2 h at 37°C, the number of Golgi profiles has doubled and the population is dominated by Berkeley bodies (60% of total). (E) Immunofluorescence microscopy using anti-Ypt1p antibodies (Segev et al., 1988). (1) wild-type cells (NSY128) grown at 26°C; (2) the ypt31-Δ/ypt32-A141D mutant (NSY348) grown at 26°C; or (3) shifted to 37°C, for 90 min; (4) the same cells shown in 3 photographed with Nomarski optics to show the contours of the cells. Bars: (A–C) 1 μm; (E) 10 μm.

Mentions: ypt31-Δ/32-A141D mutant cells display a steady state increase in the frequency of Golgi cisternae even at the permissive temperature (26°C) when compared to wild-type cells (Fig. 8 A, B, and D). This result indicates that the kinetic defect in protein transport seen in the mutant at the permissive temperature (Fig. 4 A) is accompanied by some accumulation of Golgi cisternae. However, a more dramatic change was observed in mutant cells shifted to the nonpermissive temperature (37°C). Under this growth condition, the total number of aberrant Golgi profiles roughly doubled, and the population became dominated by Berkeley bodies (60% of total at 37°C vs. 30% of total at 26°C; Fig. 8 D). These multilamellar structures, in which one cisterna appears to engulf another, were first observed in sec7 and sec14 mutant cells, which are defective in Golgi function (Novick et al., 1981). Mutant Golgi structures are larger than those seen in wild-type cells (385 ± 107 nm, and 298 ± 81 nm, respectively), are frequently observed to form stacks, and are occasionally swollen to ∼100 nm at their periphery (Fig. 9 B), indicating a possible defect in vesicle formation.


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 Golgi membranes at the  permissive temperature and  Berkeley bodies at the nonpermissive temperature.  Electron microscopy of (A)  wild-type (NSY128), (B)  ypt31-Δ/ypt32-A141D mutant strain (NSY348) at 26°C,  and (C) ypt31-Δ/ypt32A141D mutant strain  (NSY348) at 37°C. Arrows  indicate a single Golgi cisterna in a wild-type cell and  regions of Golgi accumulation in mutant cells. v, vacuole; n, nucleus. (D) Quantification of cisternal profiles  and Berkeley bodies in wildtype cells at 26°C and ypt31Δ/ypt32-A141D mutant  (NSY348) cells at 26° and  37°C. The percentage of the  total structures counted that  are cisternal is indicated by  the gray bar, and the percentage of Berkeley bodies is  represented by the black bar.  At the permissive temperature, the mutant shows a fivefold increase in the frequency of Golgi profiles, the  majority of which (70% of  total) are cisternal. After 2 h at 37°C, the number of Golgi profiles has doubled and the population is dominated by Berkeley bodies  (60% of total). (E) Immunofluorescence microscopy using anti-Ypt1p antibodies (Segev et al., 1988). (1) wild-type cells (NSY128)  grown at 26°C; (2) the ypt31-Δ/ypt32-A141D mutant (NSY348) grown at 26°C; or (3) shifted to 37°C, for 90 min; (4) the same cells  shown in 3 photographed with Nomarski optics to show the contours of the cells. Bars: (A–C) 1 μm; (E) 10 μm.
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Figure 8: ypt31-Δ/ypt32A141D mutant cells accumulate Golgi membranes at the permissive temperature and Berkeley bodies at the nonpermissive temperature. Electron microscopy of (A) wild-type (NSY128), (B) ypt31-Δ/ypt32-A141D mutant strain (NSY348) at 26°C, and (C) ypt31-Δ/ypt32A141D mutant strain (NSY348) at 37°C. Arrows indicate a single Golgi cisterna in a wild-type cell and regions of Golgi accumulation in mutant cells. v, vacuole; n, nucleus. (D) Quantification of cisternal profiles and Berkeley bodies in wildtype cells at 26°C and ypt31Δ/ypt32-A141D mutant (NSY348) cells at 26° and 37°C. The percentage of the total structures counted that are cisternal is indicated by the gray bar, and the percentage of Berkeley bodies is represented by the black bar. At the permissive temperature, the mutant shows a fivefold increase in the frequency of Golgi profiles, the majority of which (70% of total) are cisternal. After 2 h at 37°C, the number of Golgi profiles has doubled and the population is dominated by Berkeley bodies (60% of total). (E) Immunofluorescence microscopy using anti-Ypt1p antibodies (Segev et al., 1988). (1) wild-type cells (NSY128) grown at 26°C; (2) the ypt31-Δ/ypt32-A141D mutant (NSY348) grown at 26°C; or (3) shifted to 37°C, for 90 min; (4) the same cells shown in 3 photographed with Nomarski optics to show the contours of the cells. Bars: (A–C) 1 μm; (E) 10 μm.
Mentions: ypt31-Δ/32-A141D mutant cells display a steady state increase in the frequency of Golgi cisternae even at the permissive temperature (26°C) when compared to wild-type cells (Fig. 8 A, B, and D). This result indicates that the kinetic defect in protein transport seen in the mutant at the permissive temperature (Fig. 4 A) is accompanied by some accumulation of Golgi cisternae. However, a more dramatic change was observed in mutant cells shifted to the nonpermissive temperature (37°C). Under this growth condition, the total number of aberrant Golgi profiles roughly doubled, and the population became dominated by Berkeley bodies (60% of total at 37°C vs. 30% of total at 26°C; Fig. 8 D). These multilamellar structures, in which one cisterna appears to engulf another, were first observed in sec7 and sec14 mutant cells, which are defective in Golgi function (Novick et al., 1981). Mutant Golgi structures are larger than those seen in wild-type cells (385 ± 107 nm, and 298 ± 81 nm, respectively), are frequently observed to form stacks, and are occasionally swollen to ∼100 nm at their periphery (Fig. 9 B), indicating a possible defect in vesicle formation.

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