ABSTRACT
Recently, a pathway involving the highly choreographed recruitment of endocytic proteins to sites of clathrin/actin-mediated endocytosis has been revealed in budding yeast. Here, we investigated possible roles for candidate disassembly factors in regulation of the dynamics of the endocytic coat proteins Sla2p, Ent1p, Ent2p, Sla1p, Pan1p and End3p, each of which has mammalian homologues. Live cell imaging analysis revealed that in addition to the synaptojanin, Sjl2p, the Ark1p and Prk1p protein kinases, the putative Arf GTPase-activating protein, Gts1p and the Arf GTPase-interacting protein, Lsb5p, also arrive at endocytic sites late in the internalization pathway, consistent with roles in coat disassembly. Analysis of coat dynamics in various mutant backgrounds revealed that multiple pathways, including the ones mediated by an Arf guanosine triphosphatase and a synaptojanin, facilitate efficient disassembly of different endocytic coat proteins. In total, at least four separate processes are important for disassembly of endocytic complexes and efficient downstream trafficking of endocytic cargo.
Subject(s)
Capsid Proteins/metabolism , Endocytosis/physiology , Endosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Vesicular Transport Proteins/metabolism , ADP-Ribosylation Factors/genetics , ADP-Ribosylation Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Auxilins/genetics , Auxilins/metabolism , Capsid Proteins/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Transport/physiology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/genetics , Vesicular Transport Proteins/geneticsABSTRACT
We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis. In vivo Prk1p inhibition blocked pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p, Sla2p, Pan1p, Sla1p, and Ent1p. Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly. Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps. Upon inhibitor washout, the actin clumps rapidly disassembled, and properly polarized actin patches reappeared. Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins. Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch-related process, and propose that Prk1p negatively regulates the actin assembly-stimulating activity of endocytic proteins.
Subject(s)
Actins/metabolism , Cytoskeleton/metabolism , Endocytosis/physiology , Fungal Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Aurora Kinases , Fungal Proteins/genetics , Protein Kinase C , Protein Serine-Threonine Kinases/genetics , Pyrazoles/chemistry , Pyrimidines/chemistry , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Cell Surface/metabolism , Yeasts/genetics , Yeasts/metabolism , Yeasts/ultrastructureABSTRACT
In the budding yeast Saccharomyces cerevisiae, one of the main structural components of the cell wall is 1,3-beta-glucan produced by 1,3-beta-glucan synthase (GS). Yeast GS is composed of a putative catalytic subunit encoded by FKS1 and FKS2 and a regulatory subunit encoded by RHO1. A combination of amino acid alterations in the putative catalytic domain of Fks1p was found to result in a loss of the catalytic activity. To identify upstream regulators of 1,3-beta-glucan synthesis, we isolated multicopy suppressors of the GS mutation. We demonstrate that all of the multicopy suppressors obtained (WSC1, WSC3, MTL1, ROM2, LRE1, ZDS1, and MSB1) and the constitutively active RHO1 mutations tested restore 1,3-beta-glucan synthesis in the GS mutant. A deletion of either ROM2 or WSC1 leads to a significant defect of 1,3-beta-glucan synthesis. Analyses of the degree of Mpk1p phosphorylation revealed that among the multicopy suppressors, WSC1, ROM2, LRE1, MSB1, and MTL1 act positively on the Pkc1p-MAPK pathway, another signaling pathway regulated by Rho1p, while WSC3 and ZDS1 do not. We have also found that MID2 acts positively on Pkc1p without affecting 1,3-beta-glucan synthesis. These results suggest that distinct networks regulate the two effector proteins of Rho1p, Fks1p and Pkc1p.
