RESUMO
Point mutations in the human MYO1E gene, encoding class I myosin Myo1e, are associated with focal segmental glomerulosclerosis (FSGS), a primary kidney disorder that leads to end-stage kidney disease. In this study, we used a simple model organism, fission yeast Schizosaccharomyces pombe, to test the effects of FSGS-associated mutations on myosin activity. Fission yeast has only one class I myosin, Myo1, which is involved in actin patch assembly at the sites of endocytosis. The amino acid residues mutated in individuals with FSGS are conserved between human Myo1e and yeast Myo1, which allowed us to introduce equivalent mutations into yeast myosin and use the resulting mutant strains for functional analysis. Yeast strains expressing mutant Myo1 exhibited defects in growth and endocytosis similar to those observed in the myo1 deletion strain. These mutations also disrupted Myo1 localization to endocytic actin patches and resulted in mis-localization of Myo1 to eisosomes, linear membrane microdomains found in yeast cells. Although both mutants examined in this study exhibited loss of function, one of these mutants was also characterized by the decreased protein stability. Thus, using the yeast model system, we were able to determine that the kidney-disease-associated mutations impair myosin functional activity and have differential effects on protein stability.
Assuntos
Glomerulosclerose Segmentar e Focal/genética , Mutação/genética , Cadeias Pesadas de Miosina/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Actinas/metabolismo , Sequência de Aminoácidos , Biomarcadores/metabolismo , Endocitose , Humanos , Rim/patologia , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo I/química , Miosina Tipo I/genética , Estabilidade Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
The evolutionarily conserved small actin-monomer binding protein profilin is believed to be a housekeeping factor that maintains a general pool of unassembled actin. However, despite similar primary sequences, structural folds, and affinities for G-actin and poly-L-proline, budding yeast profilin ScPFY fails to complement fission yeast profilin SpPRF temperature-sensitive mutant cdc3-124 cells. To identify profilin's essential properties, we built a combinatorial library of ScPFY variants containing either WT or SpPRF residues at multiple positions and carried out a genetic selection to isolate variants that support life in fission yeast. We subsequently engineered ScPFY(9-Mut), a variant containing nine substitutions in the actin-binding region, which complements cdc3-124 cells. ScPFY(9-Mut), but not WT ScPFY, suppresses severe cytokinesis defects in cdc3-124 cells. Furthermore, the major activity rescued by ScPFY(9-Mut) is the ability to enhance cytokinesis formin Cdc12-mediated actin assembly in vitro, which allows cells to assemble functional contractile rings. Therefore an essential role of profilin is to specifically facilitate formin-mediated actin assembly for cytokinesis in fission yeast.
Assuntos
Actinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Profilinas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Actinas/química , Actinas/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citocinese/genética , Proteínas do Citoesqueleto/genética , Teste de Complementação Genética , Immunoblotting , Microscopia de Fluorescência , Modelos Moleculares , Mutação , Profilinas/química , Profilinas/genética , Prolina/química , Prolina/genética , Prolina/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Imagem com Lapso de Tempo/métodosRESUMO
Fission yeast cells use Arp2/3 complex and formin to assemble diverse filamentous actin (F-actin) networks within a common cytoplasm for endocytosis, division, and polarization. Although these homeostatic F-actin networks are usually investigated separately, competition for a limited pool of actin monomers (G-actin) helps to regulate their size and density. However, the mechanism by which G-actin is correctly distributed between rival F-actin networks is not clear. Using a combination of cell biological approaches and in vitro reconstitution of competition between actin assembly factors, we found that the small G-actin binding protein profilin directly inhibits Arp2/3 complex-mediated actin assembly. Profilin is therefore required for formin to compete effectively with excess Arp2/3 complex for limited G-actin and to assemble F-actin for contractile ring formation in dividing cells.
