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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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Details of Golgi structures in wild-type and ypt31-Δ/ypt32-A141D mutant cells. (A) Electron microscopy of wild-type  (NSY128) cells. Wild-type Golgi is usually observed as an elongated or cup-shaped single cisternae (Preuss et al., 1992), which are not  always continuous (third panel from left) because of fenestration (Rambourg et al., 1993). (B) Enlarged ypt31-Δ/ypt32-A141D mutant  (NSY348) Golgi structures. Long arrows indicate an example of stacked cisternae in the mutant strain. Short arrows indicate spherical  and tear drop–shaped Berkeley bodies. The tear-shaped structure suggests that Berkeley bodies are derived from individual cup-shaped  cisternae that fuse at the rim (see curved arrow for a possible intermediate in this process). Open arrow indicates swelling to 100 nm at  the cisternal rim, which may represent an intermediate preceding vesicle formation by membrane fission. Arrowheads indicate rare 100nm vesicles seen in the vicinity of cisternae, for size comparison with structures that look like budding vesicles. Note also the increased  length of cisternal profiles in the mutant strain compared to wild-type. Regions of four different cells are shown for each strain. Bars, 0.5 μm.
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Figure 9: Details of Golgi structures in wild-type and ypt31-Δ/ypt32-A141D mutant cells. (A) Electron microscopy of wild-type (NSY128) cells. Wild-type Golgi is usually observed as an elongated or cup-shaped single cisternae (Preuss et al., 1992), which are not always continuous (third panel from left) because of fenestration (Rambourg et al., 1993). (B) Enlarged ypt31-Δ/ypt32-A141D mutant (NSY348) Golgi structures. Long arrows indicate an example of stacked cisternae in the mutant strain. Short arrows indicate spherical and tear drop–shaped Berkeley bodies. The tear-shaped structure suggests that Berkeley bodies are derived from individual cup-shaped cisternae that fuse at the rim (see curved arrow for a possible intermediate in this process). Open arrow indicates swelling to 100 nm at the cisternal rim, which may represent an intermediate preceding vesicle formation by membrane fission. Arrowheads indicate rare 100nm vesicles seen in the vicinity of cisternae, for size comparison with structures that look like budding vesicles. Note also the increased length of cisternal profiles in the mutant strain compared to wild-type. Regions of four different cells are shown for each strain. Bars, 0.5 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)

Details of Golgi structures in wild-type and ypt31-Δ/ypt32-A141D mutant cells. (A) Electron microscopy of wild-type  (NSY128) cells. Wild-type Golgi is usually observed as an elongated or cup-shaped single cisternae (Preuss et al., 1992), which are not  always continuous (third panel from left) because of fenestration (Rambourg et al., 1993). (B) Enlarged ypt31-Δ/ypt32-A141D mutant  (NSY348) Golgi structures. Long arrows indicate an example of stacked cisternae in the mutant strain. Short arrows indicate spherical  and tear drop–shaped Berkeley bodies. The tear-shaped structure suggests that Berkeley bodies are derived from individual cup-shaped  cisternae that fuse at the rim (see curved arrow for a possible intermediate in this process). Open arrow indicates swelling to 100 nm at  the cisternal rim, which may represent an intermediate preceding vesicle formation by membrane fission. Arrowheads indicate rare 100nm vesicles seen in the vicinity of cisternae, for size comparison with structures that look like budding vesicles. Note also the increased  length of cisternal profiles in the mutant strain compared to wild-type. Regions of four different cells are shown for each strain. Bars, 0.5 μm.
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Figure 9: Details of Golgi structures in wild-type and ypt31-Δ/ypt32-A141D mutant cells. (A) Electron microscopy of wild-type (NSY128) cells. Wild-type Golgi is usually observed as an elongated or cup-shaped single cisternae (Preuss et al., 1992), which are not always continuous (third panel from left) because of fenestration (Rambourg et al., 1993). (B) Enlarged ypt31-Δ/ypt32-A141D mutant (NSY348) Golgi structures. Long arrows indicate an example of stacked cisternae in the mutant strain. Short arrows indicate spherical and tear drop–shaped Berkeley bodies. The tear-shaped structure suggests that Berkeley bodies are derived from individual cup-shaped cisternae that fuse at the rim (see curved arrow for a possible intermediate in this process). Open arrow indicates swelling to 100 nm at the cisternal rim, which may represent an intermediate preceding vesicle formation by membrane fission. Arrowheads indicate rare 100nm vesicles seen in the vicinity of cisternae, for size comparison with structures that look like budding vesicles. Note also the increased length of cisternal profiles in the mutant strain compared to wild-type. Regions of four different cells are shown for each strain. Bars, 0.5 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