Saccharomyces cerevisiae translational activator Cbs1p is associated with translationally active mitochondrial ribosomes.
In the yeast Saccharomyces cerevisiae, mitochondrial translation of most, if not all, mitochondrially encoded genes is regulated by an individual set of gene-specific activators. Translation of the COB mRNA encoding cytochrome b requires the function of two nuclearly encoded proteins, Cbs1p and Cbs2p. Genetic data revealed that the 5'-untranslated region of COB mRNA is the target of both proteins. Recently, we provided evidence for an interaction of Cbs2p with mitochondrial ribosomes. We demonstrate here by means of blue native gel electrophoresis, density gradient centrifugation and tandem affinity purification that a portion of Cbs1p is also associated with mitochondrial ribosomes. In addition, we demonstrate that the amount of ribosome-associated Cbs1p is elevated in the presence of chloramphenicol, which is known to stall ribosomes on mRNAs. In the presence of puromycin, which strips off the mRNA and nascent protein chains from ribosomes, Cbs1p is no longer associated with ribosomes. Our data indicate that the observed interaction is mediated by ribosome-bound mRNA, thus restricting the association to ribosomes actively translating cytochrome b.
Krause-Buchholz
Udo
U
Institut für Genetik, Technische Universität Dresden, D-01062 Dresden, Germany. ukrause@rcs.urz.tu-dresden.de
Schöbel
Karina
K
Lauffer
Susann
S
Rödel
Gerhard
G
D002499:Centrifugation, Density Gradient; D005591:Chemical Fractionation; D002701:Chloramphenicol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
C060724:CBS1 protein, S cerevisiae; D024101:Mitochondrial Proteins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; C060587:YMR31 protein, S cerevisiae; D011691:Puromycin; D002701:Chloramphenicol