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Purification of actin from fission yeast Schizosaccharomyces pombe and characterization of functional differences from muscle actin.

ABSTRACT: Fission yeast Schizosaccharomyces pombe is an important genetic model organism for studying the mechanisms of endocytosis and cytokinesis. However, most work on the biochemical properties of fission yeast actin-binding proteins has been done with skeletal muscle actin for matters of convenience. When simulations of mathematical models of the mechanism of endocytosis were compared with events in live cells, some of the reactions appeared to be much faster than observed in biochemical experiments with muscle actin. Here, we used gelsolin affinity chromatography to purify actin from fission yeast. S. pombe actin shares many properties with skeletal muscle actin but has higher intrinsic nucleotide exchange rate, faster trimer nucleus formation, faster phosphate dissociation rate from polymerized actin, and faster nucleation of actin filaments with Arp2/3 complex. These properties close the gap between the biochemistry and predictions made by mathematical models of endocytosis in S. pombe cells.

SUBMITTER: Ti SC

PROVIDER: S-EPMC3037691 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Purification of actin from fission yeast Schizosaccharomyces pombe and characterization of functional differences from muscle actin.

Ti Shih-Chieh SC Pollard Thomas D TD

The Journal of biological chemistry 20101209 7

Fission yeast Schizosaccharomyces pombe is an important genetic model organism for studying the mechanisms of endocytosis and cytokinesis. However, most work on the biochemical properties of fission yeast actin-binding proteins has been done with skeletal muscle actin for matters of convenience. When simulations of mathematical models of the mechanism of endocytosis were compared with events in live cells, some of the reactions appeared to be much faster than observed in biochemical experiments ...[more]

PMID: 21148484

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Project description:BackgroundHIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability.ResultsA fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8.ConclusionsThis is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.

Dataset Information

Purification of actin from fission yeast Schizosaccharomyces pombe and characterization of functional differences from muscle actin.

Publications

Purification of actin from fission yeast Schizosaccharomyces pombe and characterization of functional differences from muscle actin.

Similar Datasets

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