ABSTRACT
The BAR adaptor proteins encoded by the RVS167 and RVS161 genes from Saccharomyces cerevisiae form a complex that regulates actin, endocytosis, and viability following starvation or osmotic stress. In this study, we identified a human homolog of RVS161, termed BIN3 (bridging integrator-3), and a Schizosaccharomyces pombe homolog of RVS161, termed hob3+ (homolog of Bin3). In human tissues, the BIN3 gene was expressed ubiquitously except for brain. S. pombe cells lacking Hob3p were often multinucleate and characterized by increased amounts of calcofluor-stained material and mislocalized F-actin. For example, while wild-type cells localized F-actin to cell ends during interphase, hob3Delta mutants had F-actin patches distributed randomly around the cell. In addition, medial F-actin rings were rarely found in hob3Delta mutants. Notably, in contrast to S. cerevisiae rvs161Delta mutants, hob3Delta mutants showed no measurable defects in endocytosis or response to osmotic stress, yet hob3+ complemented the osmosensitivity of a rvs161Delta mutant. BIN3 failed to rescue the osmosensitivity of rvs161Delta, but the actin localization defects of hob3Delta mutants were completely rescued by BIN3 and partially rescued by RVS161. These findings suggest that hob3+ and BIN3 regulate F-actin localization, like RVS161, but that other roles for this gene have diverged somewhat during evolution.
Subject(s)
Actins/metabolism , Cytoskeletal Proteins , Fungal Proteins/genetics , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Saccharomyces cerevisiae Proteins , Schizosaccharomyces/genetics , Transcription, Genetic , Actins/analysis , Amino Acid Sequence , Brain/metabolism , Cell Line , DNA, Complementary , Fungal Proteins/chemistry , Gene Library , Genetic Complementation Test , Humans , Kinetics , Microfilament Proteins/chemistry , Molecular Sequence Data , Organ Specificity , RNA, Messenger/genetics , Schizosaccharomyces/growth & development , Schizosaccharomyces/metabolism , Sequence Alignment , Sequence Deletion , Sequence Homology, Amino AcidABSTRACT
At least eight conserved motifs are visible in the totivirus RNA-dependent RNA polymerase (RDRP). We have systematically altered each of these in the Saccharomyces cerevisiae double-stranded RNA virus ScVL1 by substituting the conserved motifs from a giardiavirus. The results help define the conserved regions of the RDRP involved in polymerase function and those essential for other reasons.
