Herman, Paul Kenneth (1991) Protein sorting in the eukaryotic secretory pathway : an essential role for a novel yeast protein kinase. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-06202007-083735
The yeast vps mutants are defective for the intracellular sorting of proteins to the vacuolar compartment. Mutants from two particular vps complementation groups, vpsl5 and vps34, share a common set of phenotypes that suggested that the VPS15 and VPS34 gene products might be functioning at a similar step of the vacuolar protein sorting pathway. vpsl5 and vps34 mutants exhibit specific defects in the sorting of soluble hydrolases to the vacuolar compartment. Whereas soluble hydrolases such as carboxypeptidase Y are almost quantitatively mislocalized to the cell surface, vacuolar membrane proteins appear to be properly localized to the vacuole.
The wild-type VPS15 and VPS34 genes were both cloned from yeast genomic DNA libraries by complementation of temperature-sensitive growth defects associated with mutations in these genes. Haploid yeast strains carrying a disruption of either locus were viable but exhibited a severe ts growth defect indicating that both genes are essential for vegetative growth at elevated temperatures. The vps34 null mutant was also found to exhibit a defect in the segregation of the vacuolar compartment upon cell division.
The predicted sequence of the VPS15 gene product exhibits significant similarity to the catalytic domains of the serine/threonine family of protein kinases. Point mutations altering specific amino acid residues of Vpsl5p that are highly conserved in all protein kinases result in the biological inactivation of Vpsl5p. The kinase domain mutants exhibit severe vacuolar protein sorting and ts growth defects. In addition, Vpsl5p is phosphorylated in vivo in a reaction that requires a wild-type Vpsl5p kinase domain. Subcellular fractionation experiments indicate that Vpsl5p is peripherally associated with the cytoplasmic face of a late Golgi or vesicle compartment. A vpsl5 mutant that encodes a protein lacking 30 carboxy-terminal amino acids exhibits a severe ts defect in vacuolar protein delivery. At the restrictive temperature, carboxpeptidase Y accumulates in a specific intracellular compartment that may represent a normal transport intermediate between the Golgi and vacuolar compartments. The vacuolar delivery defect in this mutant has an extremely rapid rate of onset suggesting that Vps15p is directly involved in the sorting of soluble proteins to the vacuole. Altogether, these data suggest that Vpsl5p regulates specific protein phosphorylation reactions in vivo that are required for the delivery of soluble hydrolases to the vacuole.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||23 April 1991|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||12 Jul 2007|
|Last Modified:||26 Dec 2012 02:53|
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