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Research paper thumbnail of GWT1 Gene Is Required for Inositol Acylation of Glycosylphosphatidylinositol Anchors in Yeast

Journal of Biological Chemistry, 2003

Glycosylphosphatidylinositol (GPI) is a conserved posttranslational modification to anchor cell s... more Glycosylphosphatidylinositol (GPI) is a conserved posttranslational modification to anchor cell surface proteins to plasma membrane in all eukaryotes. In yeast, GPI mediates cross-linking of cell wall mannoproteins to ␤1,6glucan. We reported previously that the GWT1 gene product is a target of the novel anti-fungal compound, 1-[4butylbenzyl]isoquinoline, that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharo

Research paper thumbnail of Erg25 Controls Host-Cholesterol Uptake Mediated by Aus1p-Associated Sterol-Rich Membrane Domains in Candida glabrata

Frontiers in Cell and Developmental Biology

The uptake of cholesterol from the host is closely linked to the proliferation of pathogenic fung... more The uptake of cholesterol from the host is closely linked to the proliferation of pathogenic fungi and protozoa during infection. For some pathogenic fungi, cholesterol uptake is an important strategy for decreasing susceptibility to antifungals that inhibit ergosterol biosynthesis. In this study, we show that Candida glabrata ERG25, which encodes an enzyme that demethylates 4,4-dimethylzymosterol, is required for cholesterol uptake from host serum. Based on the screening of C. glabrata conditional knockdown mutants for each gene involved in ergosterol biosynthesis, ERG25 knockdown was found to decrease lethality of infected mice. ERG25 knockdown impairs the plasma membrane localization of the sterol importer Aus1p, suggesting that the accumulated 4,4-dimethylzymosterol destabilizes the lipid domain with which Aus1p functionally associates. ERG25 knockdown further influences the structure of the membrane compartment of Can1p (MCC)/eisosomes (ergosterol-rich lipid domains), but not t...

Research paper thumbnail of Cooperation between ER stress and calcineurin signaling contributes to the maintenance of cell wall integrity in Candida glabrata

Fungal Biology

Candida glabrata is the second most common source of Candida infections in humans. In this pathog... more Candida glabrata is the second most common source of Candida infections in humans. In this pathogen, the maintenance of cell wall integrity (CWI) frequently precludes effective pharmacological treatment by antifungal agents. In numerous fungi, cell wall modulation is reported to be controlled by endoplasmic reticulum (ER) stress, but how the latter affects CWI maintenance in C. glabrata is not clearly understood. Here, we characterized a C. glabrata strain harboring a mutation in the CNE1 gene, which encodes a molecular chaperone associated with nascent glycoprotein maturation in the ER. Disruption of cne1 induced ER stress and caused changes in the normal cell wall structure, specifically a reduction in the β-1,6-glucan content and accumulation of chitin. Conversely, a treatment with the typical ER stress inducer tunicamycin up-regulated the production of cell wall chitin but did not affect β-1,6-glucan content. Our results also indicated that C. glabrata features a uniquely evolved ER stress-mediated CWI pathway, which differs from that in the closely related species Saccharomyces cerevisiae. Furthermore, we demonstrated that ER stress-mediated CWI pathway in C. glabrata is also induced by the disruption of other genes encoding proteins that function in a correlated manner in the quality control of N-linked glycoproteins in the ER. These results suggest that calcineurin and ER quality control system act as a platform for maintaining CWI in C. glabrata.

Research paper thumbnail of Contact of cis-Golgi with ER exit sites executes cargo capture and delivery from the ER

Nature Communications, Apr 14, 2014

Protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus is mediated by coat ... more Protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus is mediated by coat complex II (COPII) vesicles. It has been believed that COPII vesicles containing cargo are released from the ER exit sites (ERES) into the cytosol and then reach and fuse with the first post-ER compartment, cis-Golgi or ER-to-Golgi intermediate compartment (ERGIC). However, it still remains elusive how cargo loading to vesicles, vesicle budding, tethering and fusion are coordinated in vivo. Here we show, using extremely high speed and high resolution confocal microscopy, that the cis-Golgi in the budding yeast Saccharomyces cerevisiae approaches and contacts the ERES. The COPII coat cage then collapses and the cis-Golgi captures cargo. The cis-Golgi, thus loaded with cargo, then leaves the ERES. We propose that this 'hug-and-kiss' behaviour of cis-Golgi ensures efficient and targeted cargo transport from the ERES to cis-Golgi.

Research paper thumbnail of High-curvature domains of the ER are important for the organization of ER exit sites in Saccharomyces cerevisiae

Journal of Cell Science, 2012

Protein export from the endoplasmic reticulum (ER) to the Golgi apparatus occurs at specialized r... more Protein export from the endoplasmic reticulum (ER) to the Golgi apparatus occurs at specialized regions known as the ER exit sites (ERES). In Saccharomyces cerevisiae, ERES appear as numerous scattered puncta throughout the ER. We examined ERES within the peripheral ER, finding that the proteins comprising the ERES localize on high-curvature ER domains where curvature-stabilizing protein Rtn1 is present. Drtn1 Drtn2 Dyop1 cells have fewer high-curvature ER domains, but ERES accumulate at the remaining high-curvature ER domains on the edge of expanded ER sheets. We propose that membrane curvature is a key geometric feature for the regulation of ERES localization. We also investigated a spatial relationship between ERES and Golgi cisternae. Golgi cisternae in S. cerevisiae are unstacked, dispersed, and moving in the cytoplasm with cis-cisternae positioned adjacent to ERES, whereas trans-cisternae are not. Morphological changes in the ER of Drtn1 Drtn2 Dyop1 cells resulted in aberrant Golgi structures, including cis-and trans-markers, and there was reduced movement at ERES between expanded ER sheets and the plasma membrane.

Research paper thumbnail of GWT1 Gene Is Required for Inositol Acylation of Glycosylphosphatidylinositol Anchors in Yeast

Journal of Biological Chemistry, 2003

Glycosylphosphatidylinositol (GPI) is a conserved posttranslational modification to anchor cell s... more Glycosylphosphatidylinositol (GPI) is a conserved posttranslational modification to anchor cell surface proteins to plasma membrane in all eukaryotes. In yeast, GPI mediates cross-linking of cell wall mannoproteins to ␤1,6glucan. We reported previously that the GWT1 gene product is a target of the novel anti-fungal compound, 1-[4butylbenzyl]isoquinoline, that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharo

Research paper thumbnail of Erg25 Controls Host-Cholesterol Uptake Mediated by Aus1p-Associated Sterol-Rich Membrane Domains in Candida glabrata

Frontiers in Cell and Developmental Biology

The uptake of cholesterol from the host is closely linked to the proliferation of pathogenic fung... more The uptake of cholesterol from the host is closely linked to the proliferation of pathogenic fungi and protozoa during infection. For some pathogenic fungi, cholesterol uptake is an important strategy for decreasing susceptibility to antifungals that inhibit ergosterol biosynthesis. In this study, we show that Candida glabrata ERG25, which encodes an enzyme that demethylates 4,4-dimethylzymosterol, is required for cholesterol uptake from host serum. Based on the screening of C. glabrata conditional knockdown mutants for each gene involved in ergosterol biosynthesis, ERG25 knockdown was found to decrease lethality of infected mice. ERG25 knockdown impairs the plasma membrane localization of the sterol importer Aus1p, suggesting that the accumulated 4,4-dimethylzymosterol destabilizes the lipid domain with which Aus1p functionally associates. ERG25 knockdown further influences the structure of the membrane compartment of Can1p (MCC)/eisosomes (ergosterol-rich lipid domains), but not t...

Research paper thumbnail of Cooperation between ER stress and calcineurin signaling contributes to the maintenance of cell wall integrity in Candida glabrata

Fungal Biology

Candida glabrata is the second most common source of Candida infections in humans. In this pathog... more Candida glabrata is the second most common source of Candida infections in humans. In this pathogen, the maintenance of cell wall integrity (CWI) frequently precludes effective pharmacological treatment by antifungal agents. In numerous fungi, cell wall modulation is reported to be controlled by endoplasmic reticulum (ER) stress, but how the latter affects CWI maintenance in C. glabrata is not clearly understood. Here, we characterized a C. glabrata strain harboring a mutation in the CNE1 gene, which encodes a molecular chaperone associated with nascent glycoprotein maturation in the ER. Disruption of cne1 induced ER stress and caused changes in the normal cell wall structure, specifically a reduction in the β-1,6-glucan content and accumulation of chitin. Conversely, a treatment with the typical ER stress inducer tunicamycin up-regulated the production of cell wall chitin but did not affect β-1,6-glucan content. Our results also indicated that C. glabrata features a uniquely evolved ER stress-mediated CWI pathway, which differs from that in the closely related species Saccharomyces cerevisiae. Furthermore, we demonstrated that ER stress-mediated CWI pathway in C. glabrata is also induced by the disruption of other genes encoding proteins that function in a correlated manner in the quality control of N-linked glycoproteins in the ER. These results suggest that calcineurin and ER quality control system act as a platform for maintaining CWI in C. glabrata.

Research paper thumbnail of Contact of cis-Golgi with ER exit sites executes cargo capture and delivery from the ER

Nature Communications, Apr 14, 2014

Protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus is mediated by coat ... more Protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus is mediated by coat complex II (COPII) vesicles. It has been believed that COPII vesicles containing cargo are released from the ER exit sites (ERES) into the cytosol and then reach and fuse with the first post-ER compartment, cis-Golgi or ER-to-Golgi intermediate compartment (ERGIC). However, it still remains elusive how cargo loading to vesicles, vesicle budding, tethering and fusion are coordinated in vivo. Here we show, using extremely high speed and high resolution confocal microscopy, that the cis-Golgi in the budding yeast Saccharomyces cerevisiae approaches and contacts the ERES. The COPII coat cage then collapses and the cis-Golgi captures cargo. The cis-Golgi, thus loaded with cargo, then leaves the ERES. We propose that this 'hug-and-kiss' behaviour of cis-Golgi ensures efficient and targeted cargo transport from the ERES to cis-Golgi.

Research paper thumbnail of High-curvature domains of the ER are important for the organization of ER exit sites in Saccharomyces cerevisiae

Journal of Cell Science, 2012

Protein export from the endoplasmic reticulum (ER) to the Golgi apparatus occurs at specialized r... more Protein export from the endoplasmic reticulum (ER) to the Golgi apparatus occurs at specialized regions known as the ER exit sites (ERES). In Saccharomyces cerevisiae, ERES appear as numerous scattered puncta throughout the ER. We examined ERES within the peripheral ER, finding that the proteins comprising the ERES localize on high-curvature ER domains where curvature-stabilizing protein Rtn1 is present. Drtn1 Drtn2 Dyop1 cells have fewer high-curvature ER domains, but ERES accumulate at the remaining high-curvature ER domains on the edge of expanded ER sheets. We propose that membrane curvature is a key geometric feature for the regulation of ERES localization. We also investigated a spatial relationship between ERES and Golgi cisternae. Golgi cisternae in S. cerevisiae are unstacked, dispersed, and moving in the cytoplasm with cis-cisternae positioned adjacent to ERES, whereas trans-cisternae are not. Morphological changes in the ER of Drtn1 Drtn2 Dyop1 cells resulted in aberrant Golgi structures, including cis-and trans-markers, and there was reduced movement at ERES between expanded ER sheets and the plasma membrane.