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Genetic interactions between a phospholipase A2 and the Rim101 pathway components in S. cerevisiae reveal a role for this pathway in response to changes in membrane composition and shape.

Mattiazzi M, Jambhekar A, Kaferle P, Derisi JL, Krizaj I, Petrovic U - Mol. Genet. Genomics (2010)

Bottom Line: We have conducted a genome-wide analysis of cellular effects of a PLA(2) in the yeast Saccharomyces cerevisiae as a model system.Our results suggest that the composition and/or the shape of the endosomal membrane affect the Rim101 pathway.This study provides a list of the players involved in the global response to changes in membrane composition and shape in a model eukaryotic cell, and further studies are needed to understand the precise molecular mechanisms connecting them.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Biomedical Sciences, Jozef Stefan Institute, 1000 Ljubljana, Slovenia.

ABSTRACT
Modulating composition and shape of biological membranes is an emerging mode of regulation of cellular processes. We investigated the global effects that such perturbations have on a model eukaryotic cell. Phospholipases A(2) (PLA(2)s), enzymes that cleave one fatty acid molecule from membrane phospholipids, exert their biological activities through affecting both membrane composition and shape. We have conducted a genome-wide analysis of cellular effects of a PLA(2) in the yeast Saccharomyces cerevisiae as a model system. We demonstrate functional genetic and biochemical interactions between PLA(2) activity and the Rim101 signaling pathway in S. cerevisiae. Our results suggest that the composition and/or the shape of the endosomal membrane affect the Rim101 pathway. We describe a genetically and functionally related network, consisting of components of the Rim101 pathway and the prefoldin, retromer and SWR1 complexes, and predict its functional relation to PLA(2) activity in a model eukaryotic cell. This study provides a list of the players involved in the global response to changes in membrane composition and shape in a model eukaryotic cell, and further studies are needed to understand the precise molecular mechanisms connecting them.

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Western blot showing the abundance of Rim101p and its proteolytic cleavage products in the presence and absence of PLA2 at different pH values. The RIM101-HA-tagged WT (wild-type strain with empty vector) and RIM101-HA-tagged PLA2-expressing strain (wild-type strain with PLA2-expressing vector) were grown overnight in YNBglc + CSM-Ura medium and then diluted in identical medium buffered to pH 3.5, 4.5 or 7.5. The cells were collected in mid-log phase and the assay was carried out as described in “Materials and methods”. Pgk1 was used as a loading control. Full-length and cleaved Rim101p are indicated. In the PLA2-expressing strain, the ratio of cleaved to non-cleaved Rim101p under all experimental conditions is normal. The abundance of Rim101p at alkaline pH is unchanged. At acidic pH, on the other hand, significantly more Rim101p is present in PLA2-expressing cells
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Fig1: Western blot showing the abundance of Rim101p and its proteolytic cleavage products in the presence and absence of PLA2 at different pH values. The RIM101-HA-tagged WT (wild-type strain with empty vector) and RIM101-HA-tagged PLA2-expressing strain (wild-type strain with PLA2-expressing vector) were grown overnight in YNBglc + CSM-Ura medium and then diluted in identical medium buffered to pH 3.5, 4.5 or 7.5. The cells were collected in mid-log phase and the assay was carried out as described in “Materials and methods”. Pgk1 was used as a loading control. Full-length and cleaved Rim101p are indicated. In the PLA2-expressing strain, the ratio of cleaved to non-cleaved Rim101p under all experimental conditions is normal. The abundance of Rim101p at alkaline pH is unchanged. At acidic pH, on the other hand, significantly more Rim101p is present in PLA2-expressing cells

Mentions: For the induction of the Rim101 pathway under alkaline pH, Rim101p must be cleaved by the protease Rim13p (Futai et al. 1999). To investigate further the relationship between PLA2 activity and the Rim101 pathway, we analyzed the abundance of Rim101p and its proteolytic cleavage pattern in the presence and absence of PLA2 at different pH values (Fig. 1). At each pH, we observed the same fraction of cleaved Rim101p in the wild type as compared to PLA2-expressing cells. All Rim101p was cleaved at basic pH in both strains and was present in similar amounts. At acidic pH, however, significantly more total Rim101p was present in the PLA2-expressing cells, although the ratio of full-length to cleaved protein appeared similar to that in the wild-type cells. Thus, PLA2 activity increased the abundance of the active (i.e., cleaved) form of Rim101p at acidic pH, where PLA2 expression does not affect the growth of yeast cells (see above).Fig. 1


Genetic interactions between a phospholipase A2 and the Rim101 pathway components in S. cerevisiae reveal a role for this pathway in response to changes in membrane composition and shape.

Mattiazzi M, Jambhekar A, Kaferle P, Derisi JL, Krizaj I, Petrovic U - Mol. Genet. Genomics (2010)

Western blot showing the abundance of Rim101p and its proteolytic cleavage products in the presence and absence of PLA2 at different pH values. The RIM101-HA-tagged WT (wild-type strain with empty vector) and RIM101-HA-tagged PLA2-expressing strain (wild-type strain with PLA2-expressing vector) were grown overnight in YNBglc + CSM-Ura medium and then diluted in identical medium buffered to pH 3.5, 4.5 or 7.5. The cells were collected in mid-log phase and the assay was carried out as described in “Materials and methods”. Pgk1 was used as a loading control. Full-length and cleaved Rim101p are indicated. In the PLA2-expressing strain, the ratio of cleaved to non-cleaved Rim101p under all experimental conditions is normal. The abundance of Rim101p at alkaline pH is unchanged. At acidic pH, on the other hand, significantly more Rim101p is present in PLA2-expressing cells
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2872012&req=5

Fig1: Western blot showing the abundance of Rim101p and its proteolytic cleavage products in the presence and absence of PLA2 at different pH values. The RIM101-HA-tagged WT (wild-type strain with empty vector) and RIM101-HA-tagged PLA2-expressing strain (wild-type strain with PLA2-expressing vector) were grown overnight in YNBglc + CSM-Ura medium and then diluted in identical medium buffered to pH 3.5, 4.5 or 7.5. The cells were collected in mid-log phase and the assay was carried out as described in “Materials and methods”. Pgk1 was used as a loading control. Full-length and cleaved Rim101p are indicated. In the PLA2-expressing strain, the ratio of cleaved to non-cleaved Rim101p under all experimental conditions is normal. The abundance of Rim101p at alkaline pH is unchanged. At acidic pH, on the other hand, significantly more Rim101p is present in PLA2-expressing cells
Mentions: For the induction of the Rim101 pathway under alkaline pH, Rim101p must be cleaved by the protease Rim13p (Futai et al. 1999). To investigate further the relationship between PLA2 activity and the Rim101 pathway, we analyzed the abundance of Rim101p and its proteolytic cleavage pattern in the presence and absence of PLA2 at different pH values (Fig. 1). At each pH, we observed the same fraction of cleaved Rim101p in the wild type as compared to PLA2-expressing cells. All Rim101p was cleaved at basic pH in both strains and was present in similar amounts. At acidic pH, however, significantly more total Rim101p was present in the PLA2-expressing cells, although the ratio of full-length to cleaved protein appeared similar to that in the wild-type cells. Thus, PLA2 activity increased the abundance of the active (i.e., cleaved) form of Rim101p at acidic pH, where PLA2 expression does not affect the growth of yeast cells (see above).Fig. 1

Bottom Line: We have conducted a genome-wide analysis of cellular effects of a PLA(2) in the yeast Saccharomyces cerevisiae as a model system.Our results suggest that the composition and/or the shape of the endosomal membrane affect the Rim101 pathway.This study provides a list of the players involved in the global response to changes in membrane composition and shape in a model eukaryotic cell, and further studies are needed to understand the precise molecular mechanisms connecting them.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Biomedical Sciences, Jozef Stefan Institute, 1000 Ljubljana, Slovenia.

ABSTRACT
Modulating composition and shape of biological membranes is an emerging mode of regulation of cellular processes. We investigated the global effects that such perturbations have on a model eukaryotic cell. Phospholipases A(2) (PLA(2)s), enzymes that cleave one fatty acid molecule from membrane phospholipids, exert their biological activities through affecting both membrane composition and shape. We have conducted a genome-wide analysis of cellular effects of a PLA(2) in the yeast Saccharomyces cerevisiae as a model system. We demonstrate functional genetic and biochemical interactions between PLA(2) activity and the Rim101 signaling pathway in S. cerevisiae. Our results suggest that the composition and/or the shape of the endosomal membrane affect the Rim101 pathway. We describe a genetically and functionally related network, consisting of components of the Rim101 pathway and the prefoldin, retromer and SWR1 complexes, and predict its functional relation to PLA(2) activity in a model eukaryotic cell. This study provides a list of the players involved in the global response to changes in membrane composition and shape in a model eukaryotic cell, and further studies are needed to understand the precise molecular mechanisms connecting them.

Show MeSH
Related in: MedlinePlus