ABSTRACT
The Schizosaccharomyces pombe eIF3a ortholog (SpeIF3a) was shown to be unable to substitute for S. cerevisiae eIF3a (SceIF3a) in its essential function in the initiation of translation. Overproduction of SpeIF3a altered the distribution of SceIF3a but formation of the endogenous eIF3 complex was not affected. SpeIF3a was found to be more tightly bound to S. cerevisiae ribosomes than SceIF3a and other eIF3 subunits (eIF3g, eIF3i, eIF3j). The host cells displayed aberrant morphology and altered chitin deposition. SpeIF3a probably competes with SceIF3a for binding to either ribosomes or yet to be identified substrates.
Subject(s)
Eukaryotic Initiation Factor-3/genetics , Eukaryotic Initiation Factor-3/physiology , Protein Biosynthesis/genetics , Saccharomyces cerevisiae/growth & development , Schizosaccharomyces pombe Proteins/physiology , Schizosaccharomyces/genetics , Cloning, Molecular , Cytoplasm/chemistry , Gene Deletion , Genetic Complementation Test , Microscopy, Confocal , Microscopy, Fluorescence , Protein Binding , Ribosomes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/physiology , Schizosaccharomyces pombe Proteins/geneticsABSTRACT
The essential gene RPG1/TIF32 of Saccharomyces cerevisiae encodes the 110-kDa subunit of the translation initiation factor 3 (eIF3) core complex. In this study, the Rpg1p-specific monoclonal antibody PK1/1 was used to analyse the cellular distribution of Rpg1p by epifluorescence and confocal laser scanning microscopy (CLSM). In budded cells, a portion of Rpg1p was obviously co-localised with microtubules. In addition, CLSM revealed an accumulation of Rpg1p in a patch at the very end of cytoplasmic microtubules reaching the bud tip. A punctate fluorescence pattern was typical for separated unbudded cells. Distribution of Rpg1p was confirmed using a strain expressing exclusively a hemaglutinin-tagged version of Rpg1p. In nocodazole-treated cells, the pattern of the PK1/1 staining was disturbed. No staining was observed in Rpg1p-depleted cells. In vitro experiments revealed that Rpg1p was specifically co-immunoprecipitated with alpha-tubulin from the yeast cell free extract and this observation was further supported by showing that Rpg1p co-sedimented with hog brain microtubules. We conclude that Rpg1p is a microtubule-interacting protein that indicates an interesting connection between the translation initiation machinery and cytoskeleton in yeast Saccharomyces cerevisiae.
