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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 protein is found at sites of polarized cell growth. (A) Characterization of anti-Ypt31 antibodies. Antibodies raised  against Ypt31p react with Ypt31p, and to a lesser degree with Ypt32p, but not Ypt1 and Sec4 proteins. Bacterially purified Ypt1, Ypt31,  Ypt32, and Sec4 proteins were analyzed by SDS-PAGE followed by either Coomassie staining (1 μg protein in each lane) or Western  blotting (5 ng protein in each lane) using anti-Ypt1 or anti-Ypt31 antibodies. (B) Detection of Ypt31 and Ypt32 proteins in yeast cell extracts: Ypt31p is present in excess of Ypt32p in wild-type yeast cells. Wild-type (NSY128) or deletion strains (Δypt31, NSY 290 and  Δypt32, NSY296) transformed with the indicated plasmids were grown in synthetic medium maintaining selection for deletions and plasmids present. Cells were lysed by the addition of 2× Laemmli buffer and boiling for 5 min. 0.5 OD600 U of lysate from each strain was  then subjected to immunoblot analysis using affinity-purified anti-Ypt31 antibodies. (C) Localization of the Ypt31 protein in yeast cells  using immunofluorescence microscopy. Diploid yeast cells (JK9-3d) were double stained with rhodamine-phalloidin (to visualize filamentous actin) and with anti-Ypt31 antibodies. Cells were also photographed using Nomarski optics to distinguish budded from unbudded  cells (top). Arrowheads indicate polarized Ypt31 staining, which localizes to regions of cell growth in (from left to right) a cell in late G1,  a cell in early S phase, and a cell undergoing cytokinesis. Bar, 10 μm.
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Figure 2: Ypt31 protein is found at sites of polarized cell growth. (A) Characterization of anti-Ypt31 antibodies. Antibodies raised against Ypt31p react with Ypt31p, and to a lesser degree with Ypt32p, but not Ypt1 and Sec4 proteins. Bacterially purified Ypt1, Ypt31, Ypt32, and Sec4 proteins were analyzed by SDS-PAGE followed by either Coomassie staining (1 μg protein in each lane) or Western blotting (5 ng protein in each lane) using anti-Ypt1 or anti-Ypt31 antibodies. (B) Detection of Ypt31 and Ypt32 proteins in yeast cell extracts: Ypt31p is present in excess of Ypt32p in wild-type yeast cells. Wild-type (NSY128) or deletion strains (Δypt31, NSY 290 and Δypt32, NSY296) transformed with the indicated plasmids were grown in synthetic medium maintaining selection for deletions and plasmids present. Cells were lysed by the addition of 2× Laemmli buffer and boiling for 5 min. 0.5 OD600 U of lysate from each strain was then subjected to immunoblot analysis using affinity-purified anti-Ypt31 antibodies. (C) Localization of the Ypt31 protein in yeast cells using immunofluorescence microscopy. Diploid yeast cells (JK9-3d) were double stained with rhodamine-phalloidin (to visualize filamentous actin) and with anti-Ypt31 antibodies. Cells were also photographed using Nomarski optics to distinguish budded from unbudded cells (top). Arrowheads indicate polarized Ypt31 staining, which localizes to regions of cell growth in (from left to right) a cell in late G1, a cell in early S phase, and a cell undergoing cytokinesis. Bar, 10 μm.

Mentions: To study the intracellular expression and localization of Ypt31/32 GTPases, we purified recombinant Ypt31 and Ypt32 proteins and raised polyclonal antibodies against Ypt31p. The bacterially purified proteins both migrate on a denaturing gel with apparent molecular mass of ∼23 kD, with Ypt31p migrating somewhat more slowly than Ypt32p (Fig. 2 A, top). The antibody recognizes both bacterially produced proteins by immunoblot analysis, but its level of detection of Ypt31p is about threefold higher than that for Ypt32p. The antibodies did not react with the closest Ypt31/32p homologues, Ypt1p and Sec4p (Fig. 2 A, bottom). The affinity-purified anti-Ypt31p antibodies reveal one protein band in wild-type yeast cells. This band corresponds to Ypt31p since it is absent in cells deleted for the YPT31 gene and is more abundant in cells expressing the YPT31 gene on a 2μ high copy number plasmid. However, the antibodies also recognize Ypt32p when it is expressed from a 2μ plasmid in a YPT31- strain (Fig. 2 B). Comparing the levels of Ypt31 and Ypt32 proteins in cells expressing them from a 2μ plasmid, in a strain deleted for YPT31 gene, suggests that Ypt31 is considerably more abundant than Ypt32 in yeast cells (about 5- to 10-fold more abundant, taking into account a threefold better detection of Ypt31p by the antibody). Fractionation of yeast cells followed by immunoblot analysis revealed that ∼90% of the Ypt31p molecules are associated with the membranous fraction (100,000 g pellet; data not shown).


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 protein is found at sites of polarized cell growth. (A) Characterization of anti-Ypt31 antibodies. Antibodies raised  against Ypt31p react with Ypt31p, and to a lesser degree with Ypt32p, but not Ypt1 and Sec4 proteins. Bacterially purified Ypt1, Ypt31,  Ypt32, and Sec4 proteins were analyzed by SDS-PAGE followed by either Coomassie staining (1 μg protein in each lane) or Western  blotting (5 ng protein in each lane) using anti-Ypt1 or anti-Ypt31 antibodies. (B) Detection of Ypt31 and Ypt32 proteins in yeast cell extracts: Ypt31p is present in excess of Ypt32p in wild-type yeast cells. Wild-type (NSY128) or deletion strains (Δypt31, NSY 290 and  Δypt32, NSY296) transformed with the indicated plasmids were grown in synthetic medium maintaining selection for deletions and plasmids present. Cells were lysed by the addition of 2× Laemmli buffer and boiling for 5 min. 0.5 OD600 U of lysate from each strain was  then subjected to immunoblot analysis using affinity-purified anti-Ypt31 antibodies. (C) Localization of the Ypt31 protein in yeast cells  using immunofluorescence microscopy. Diploid yeast cells (JK9-3d) were double stained with rhodamine-phalloidin (to visualize filamentous actin) and with anti-Ypt31 antibodies. Cells were also photographed using Nomarski optics to distinguish budded from unbudded  cells (top). Arrowheads indicate polarized Ypt31 staining, which localizes to regions of cell growth in (from left to right) a cell in late G1,  a cell in early S phase, and a cell undergoing cytokinesis. Bar, 10 μm.
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Figure 2: Ypt31 protein is found at sites of polarized cell growth. (A) Characterization of anti-Ypt31 antibodies. Antibodies raised against Ypt31p react with Ypt31p, and to a lesser degree with Ypt32p, but not Ypt1 and Sec4 proteins. Bacterially purified Ypt1, Ypt31, Ypt32, and Sec4 proteins were analyzed by SDS-PAGE followed by either Coomassie staining (1 μg protein in each lane) or Western blotting (5 ng protein in each lane) using anti-Ypt1 or anti-Ypt31 antibodies. (B) Detection of Ypt31 and Ypt32 proteins in yeast cell extracts: Ypt31p is present in excess of Ypt32p in wild-type yeast cells. Wild-type (NSY128) or deletion strains (Δypt31, NSY 290 and Δypt32, NSY296) transformed with the indicated plasmids were grown in synthetic medium maintaining selection for deletions and plasmids present. Cells were lysed by the addition of 2× Laemmli buffer and boiling for 5 min. 0.5 OD600 U of lysate from each strain was then subjected to immunoblot analysis using affinity-purified anti-Ypt31 antibodies. (C) Localization of the Ypt31 protein in yeast cells using immunofluorescence microscopy. Diploid yeast cells (JK9-3d) were double stained with rhodamine-phalloidin (to visualize filamentous actin) and with anti-Ypt31 antibodies. Cells were also photographed using Nomarski optics to distinguish budded from unbudded cells (top). Arrowheads indicate polarized Ypt31 staining, which localizes to regions of cell growth in (from left to right) a cell in late G1, a cell in early S phase, and a cell undergoing cytokinesis. Bar, 10 μm.
Mentions: To study the intracellular expression and localization of Ypt31/32 GTPases, we purified recombinant Ypt31 and Ypt32 proteins and raised polyclonal antibodies against Ypt31p. The bacterially purified proteins both migrate on a denaturing gel with apparent molecular mass of ∼23 kD, with Ypt31p migrating somewhat more slowly than Ypt32p (Fig. 2 A, top). The antibody recognizes both bacterially produced proteins by immunoblot analysis, but its level of detection of Ypt31p is about threefold higher than that for Ypt32p. The antibodies did not react with the closest Ypt31/32p homologues, Ypt1p and Sec4p (Fig. 2 A, bottom). The affinity-purified anti-Ypt31p antibodies reveal one protein band in wild-type yeast cells. This band corresponds to Ypt31p since it is absent in cells deleted for the YPT31 gene and is more abundant in cells expressing the YPT31 gene on a 2μ high copy number plasmid. However, the antibodies also recognize Ypt32p when it is expressed from a 2μ plasmid in a YPT31- strain (Fig. 2 B). Comparing the levels of Ypt31 and Ypt32 proteins in cells expressing them from a 2μ plasmid, in a strain deleted for YPT31 gene, suggests that Ypt31 is considerably more abundant than Ypt32 in yeast cells (about 5- to 10-fold more abundant, taking into account a threefold better detection of Ypt31p by the antibody). Fractionation of yeast cells followed by immunoblot analysis revealed that ∼90% of the Ypt31p molecules are associated with the membranous fraction (100,000 g pellet; data not shown).

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