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B Pishvaee

Publications and source records attributed to B Pishvaee.

6 recordsLinked to original sources
PubMed2000

A yeast DNA J protein required for uncoating of clathrin-coated vesicles in vivo.

Clathrin-coated vesicles mediate diverse processes such as nutrient uptake, downregulation of hormone receptors, formation of synaptic vesicles, virus entry, and transport of biosynthetic proteins to lysosomes. Cycles of coat assembly and disassembly are integral features of clathrin-mediated vesicular transport (Fig. 1a). Coat assembly involves recruitment of clathrin triskelia, adaptor complexes and other factors that influence coat assembly, cargo sequestration, membrane invagination and scission (Fig. 1a). Coat disassembly is thought to be essential for fusion of vesicles with target membranes and for recycling components of clathrin coats to the cytoplasm for further rounds of vesicle formation. In vitro, cytosolic heat-shock protein 70 (Hsp70) and the J-domain co-chaperone auxilin catalyse coat disassembly. However, a specific function of these factors in uncoating in vivo has not been demonstrated, leaving the physiological mechanism and significance of uncoating unclear. Here we report the identification and characterization of a Saccharomyces cerevisiae J-domain protein, Aux1. Inactivation of Aux1 results in accumulation of clathrin-coated vesicles, impaired cargo delivery, and an increased ratio of vesicle-associated to cytoplasmic clathrin. Our results demonstrate an in vivo uncoating function of a J domain co-chaperone and establish the physiological significance of uncoating in transport mediated by clathrin-coated vesicles.

Adaptor Proteins, Vesicular Transport↗
PubMed1999

A model for dynamin self-assembly based on binding between three different protein domains.

Dynamin is a 100-kDa GTPase that assembles into multimeric spirals at the necks of budding clathrin-coated vesicles. We describe three different intramolecular binding interactions that may account for the process of dynamin self-assembly. The first binding interaction is the dimerization of a 100-amino acid segment in the C-terminal half of dynamin. We call this segment the assembly domain, because it appears to be critical for multimerization. The second binding interaction occurs between the assembly domain and the N-terminal GTPase domain. The strength of this interaction is controlled by the nucleotide-bound state of the GTPase domain, as shown with mutations in GTP binding motifs and in vitro binding experiments. The third binding interaction occurs between the assembly domain and a segment that we call the middle domain. This is the segment between the N-terminal GTPase domain and the pleckstrin homology domain. The three different binding interactions suggest a model in which dynamin molecules first dimerize. The dimers are then linked into a chain by a second binding reaction. The third binding interaction might connect adjacent rungs of the spiral.

Dynamins↗
PubMed1999

A modulatory role for clathrin light chain phosphorylation in Golgi membrane protein localization during vegetative growth and during the mating response of Saccharomyces cerevisiae.

The role of clathrin light chain phosphorylation in regulating clathrin function has been examined in Saccharomyces cerevisiae. The phosphorylation state of yeast clathrin light chain (Clc1p) in vivo was monitored by [32P]phosphate labeling and immunoprecipitation. Clc1p was phosphorylated in growing cells and also hyperphosphorylated upon activation of the mating response signal transduction pathway. Mating pheromone-stimulated hyperphosphorylation of Clc1p was dependent on the mating response signal transduction pathway MAP kinase Fus3p. Both basal and stimulated phosphorylation occurred exclusively on serines. Mutagenesis of Clc1p was used to map major phosphorylation sites to serines 52 and 112, but conversion of all 14 serines in Clc1p to alanines [S(all)A] was necessary to eliminate phosphorylation. Cells expressing the S(all)A mutant Clc1p displayed no defects in Clc1p binding to clathrin heavy chain, clathrin trimer stability, sorting of a soluble vacuolar protein, or receptor-mediated endocytosis of mating pheromone. However, the trans-Golgi network membrane protein Kex2p was not optimally localized in mutant cells. Furthermore, pheromone treatment exacerbated the Kex2p localization defect and caused a corresponding defect in Kex2p-mediated maturation of the alpha-factor precursor. The results reveal a novel requirement for clathrin during the mating response and suggest that phosphorylation of the light chain subunit modulates the activity of clathrin at the trans-Golgi network.

Amino Acid Sequence↗
PubMed1997

A novel structural model for regulation of clathrin function.

The distinctive triskelion shape of clathrin allows assembly into polyhedral lattices during the process of clathrin-coated vesicle formation. We have used random and site-directed mutagenesis of the yeast clathrin heavy chain gene (CHC1) to characterize regions which determine Chc trimerization and binding to the clathrin light chain (Clc) subunit. Analysis of the mutants indicates that mutations in the trimerization domain at the triskelion vertex, as well as mutations in the adjacent leg domain, frequently influence Clc binding. Strikingly, one mutation in the trimerization domain enhances the association of Clc with Chc. Additional mutations in the trimerization domain, in combination with mutations in the adjacent leg domain, exhibit severe defects in Clc binding while maintaining near normal trimerization properties. The position of these trimerization domain mutations on one face of a putative alpha-helix defines a region on the trimer surface that interacts directly with Clc. These results suggest that Clc extends into the Chc trimerization domain from the adjacent leg, thereby bridging the two domains. On the basis of this conclusion, we propose a new model for the organization of the triskelion vertex which provides a structural basis for regulatory effects of Clc on clathrin function.

Amino Acid Sequence↗
PubMed1996

The light chain subunit is required for clathrin function in Saccharomyces cerevisiae.

Clathrin, a multimeric protein involved in intracellular protein trafficking, is composed of three heavy chains (Chc) and three light chains (Clc). Upon disruption (clc1Delta) of the single Clc-encoding gene (CLC1) in yeast, the steady state protein levels of Chc decreased 5-10-fold compared with wild type cells; consequently, phenotypes exhibited by clc1Delta cells may result indirectly from the loss of Chc as opposed to the absence of Clc. As an approach to directly examine Clc function, clc1Delta strains were generated that carry a multicopy plasmid containing the clathrin heavy chain gene (CHC1), resulting in levels of Chc 5-10-fold elevated over wild-type levels. As with deletion of CHC1, deletion of CLC1 results in defects in growth, receptor-mediated endocytosis, and maturation of the mating pheromone alpha-factor. However, elevated Chc expression in clc1Delta cells partially suppresses the growth and alpha-factor maturation defects displayed by clc1Delta cells alone. Biochemical analyses indicate that trimerization and assembly of Chc are perturbed in the absence of Clc, resulting in vesiculation defects. Our results demonstrate that the light chain subunit of clathrin is required for efficient Chc trimerization, proper formation of clathrin coats, and the generation of clathrin-coated vesicles.

Biological Transport↗