In vivo protein tyrosine nitration in S. cerevisiae: identification of tyrosine-nitrated proteins in mitochondria.
Protein tyrosine nitration (PTN) is a selective post-translational modification often associated with pathophysiological conditions. Although yeast cells lack of mammalian nitric oxide synthase (NOS) orthologues, still it has been shown that they are capable of producing nitric oxide (NO). Our studies showed that NO or reactive nitrogen species (RNS) produced in flavohemoglobin mutant (Deltayhb1) strain along with the wild type strain (Y190) of Saccharomyces cerevisiae can be visualized using specific probe 4,5-diaminofluorescein diacetate (DAF-2DA). Deltayhb1 strain of S. cerevisiae showed bright fluorescence under confocal microscope that proves NO or RNS accumulation is more in absence of flavohemoglobin. We further investigated PTN profile of both cytosol and mitochondria of Y190 and Deltayhb1 cells of S. cerevisiae using two-dimensional (2D) gel electrophoresis followed by western blot analysis. Surprisingly, we observed many immunopositive spots both in cytosol and in mitochondria from Y190 and Deltayhb1 using monoclonal anti-3-nitrotyrosine antibody indicating a basal level of NO or nitrite or peroxynitrite is produced in yeast system. To identify proteins nitrated in vivo we analyzed mitochondrial proteins from Y190 strains of S. cerevisiae. Among the eight identified proteins, two target mitochondrial proteins are aconitase and isocitrate dehydrogenase that are involved directly in the citric acid cycle. This investigation is the first comprehensive study to identify mitochondrial proteins nitrated in vivo.
Bhattacharjee
Arindam
A
Department of Biochemistry, University College of Science, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700 019, India.
Majumdar
Uddalak
U
Maity
Debasis
D
Sarkar
Tuhin Subhra
TS
Goswami
Achintya Mohan
AM
Sahoo
Rupam
R
Ghosh
Sanjay
S
D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D024101:Mitochondrial Proteins; D009566:Nitrates; D029701:Saccharomyces cerevisiae Proteins; C002744:3-nitrotyrosine; D014443:Tyrosine