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Subcellular localization of Saccharomyces cerevisiae glutamate dehydrogenase isoenzymes
Author(s) -
Petersen Max,
Trotter Pamela J.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.658.6
Subject(s) - glutamate dehydrogenase , biochemistry , nad+ kinase , saccharomyces cerevisiae , mitochondrion , biology , subcellular localization , cytosol , enzyme , isozyme , hexokinase , glutamate receptor , glycolysis , yeast , gene , receptor
Reductive amination of 2‐oxoglutarate by glutamate dehydrogenase (GDH) is a primary pathway for glutamate synthesis in Saccharomyces cerevisiae. There are three GDH isoenzymes: NADP‐dependent Gdh1p and Gdh3p and NAD‐dependent Gdh2p. Knowing the subcellular localization of the GDH enzymes is vital to understanding the role of compartmentation in glutamate homeostasis. Previous studies suggested cytosolic, nuclear, and/or mitochondrial localization, but the precise location of the enzymes remains unclear. Wild‐type and mutant ( gdh3Δ ) cells were grown on lactate to induce mitochondrial biogenesis, spheroplasted, and homogenized. Then, crude mitochondrial and extramitochondrial fractions were isolated by sedimentation. Analysis of cytochrome c oxidase as a mitochondrial marker and hexokinase as a cytosolic marker revealed limited cross‐fractional contamination. Measurement of NADP‐dependent and NAD‐dependent GDH activity conclusively demonstrated extramitochondrial localization of Gdh1p and Gdh2p enzyme activity, respectively. Comparison of data from wild‐type as compared to gdh3Δ cells implies extramitochondrial localization for Gdh3p. These data indicate that in S. cerevisiae 2‐oxoglutarate generated within the mitochondria must be transported out for conversion to glutamate by GDHs. (Supported by NIH grant GM069372 [P. J. T.] and a gift from the Grilly family [M.C.P.]).