Actin structure and function: roles in mitochondrial organization and morphogenesis in budding yeast and identification of the phalloidin-binding site.

Abstract

To further elucidate the functions of actin in budding yeast and to relate actin structure to specific roles and interactions in vivo, we determined the phenotypes caused by 13 charged-to-alanine mutations isolated previously in the single Saccharomyces cerevisiae actin gene. Defects in actin organization, morphogenesis, budding pattern, chitin deposition, septation, nuclear segregation, and mitochondrial organization were observed. In wild-type cells, mitochondria were found to be aligned along actin cables. Many of the amino acid substitutions that had the most severe effects on mitochondrial organization are located under the myosin "footprint" on the actin monomer, suggesting that actin-myosin interactions might underlie mitochondrial organization in yeast. In addition, one mutant (act1-129; R177A, D179A) produced an actin that assembled into cables and patches that could be visualized by anti-actin immunofluorescence in situ and that assembled into microfilaments of normal appearance in vitro as judged by electron microscopy but which could not be labeled by rhodamine-phalloidin in situ or in vitro. Rhodamine-phalloidin could label actin filaments assembled from all of the other mutant actins, including one (act1-119; R116A, E117A, K118A) that is altered at a residue (E117) that can be chemically cross-linked to phalloidin. The implication of residues R177 and/or D179 in phalloidin binding is in close agreement with a recently reported molecular model in which the phalloidin-binding site is proposed to be at the junction of two or three actin monomers in the filament.

Functional Interaction between Phosducin-like Protein 2 and Cytosolic Chaperonin Is Essential for Cytoskeletal Protein Function and Cell Cycle Progression

“…Moreover, after 8 h of growth at 37°C, the plp2-ts cells exhibited budding defects such as multiple buds (0% wild type, 7% plp2-1, 30% plp2-2; p Ͻ 0.01; Supplemental Table S3) and a thickening of the bud-neck junction between mother and daughter cells (0% wild-type, 11% plp2-1, 26% plp2-2; p Ͻ 0.001; Supplemental Table S4). These types of budding defects are also consistent with disrupted actin function or organization (Drubin et al, 1993).…”

“…When cultured at the nonpermissive temperature of 37°C, plp2-ts cells became larger ( Figure 3B), a possible indication of defects in actin filament organization (Drubin et al, 1993). Indeed, plp2-1 cells were significantly larger than their wild-type counterparts even at permissive temperatures ( Figure 3B).…”

An actin point‐mutation neighboring the ‘hydrophobic plug’ causes defects in the maintenance of cell polaroty and septum organization in the fission yeast Schizosaccharomyces pombe

“…1H). These results suggest that eps8 F-actin filaments are still present but in an altered conformation, rather than change in phalloidin-binding site(s) itself [27], to which phalloidin cannot bind. After being shifted to restrictive conditions, multinucleate cells divide rapidly into several cells that contain one, and often, two incompletely segregated nuclei.…”

“…This, however, may not necessarily mean that no filamentous actin exists within the cell. In S. eerevisiae, an actin having two altered residues (R177A, D179A) was reported to be assembled into F-actin filaments which could be visualized by anti-actin immunofluorescence but not by phalloidin [27]. To determine whether this was the case, anti-actin monoclonal antibody staining was carried out.…”

Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

“…Either hyperstable or slowly polymerizing F-actin filaments might be expected to alter the dynamics and regulation of the late stages of endocytosis. Third, act1-121 mutant diploid cells have the relatively unique phenotype compared with many other act1 alleles of hyperpolarized and elongated buds, a phenotype also associated with mutations in septins and the p21-activated kinase homologue Cla4 (Drubin et al, 1993;Richman et al, 1999;Versele and Thorner, 2004). Cla4 also regulates Myo3 and Myo5 activity (Wu et al, 1996).…”