KIN1 and KIN2 protein kinases localize to the cytoplasmic face of the yeast plasma membrane.
Two genes, KIN1 and KIN2, were isolated in Saccharomyces cerevisiae by their homology to the protein kinase family of viral oncogenes. In previous studies, we identified the yeast KIN1 gene product (pp145KIN1) and KIN2 gene product (pp145KIN2) as 145 kDa phosphoproteins with serine/threonine protein kinase activity. We have used two approaches to determine the location of these protein kinases in the yeast cell: biochemical fractionation and purification of plasma membrane and cell wall material. Biochemical fractionation techniques revealed that 70-90% of both KIN1 and KIN2 proteins localized to a particulate fraction. This particulate fraction contained three pools of KIN1 and KIN2 proteins. One pool could be released with either sodium chloride or high pH, another released with nonionic detergent, and a third was resistant to either treatment. Thus, both protein-protein and protein-membrane interactions are responsible for the association to the particulate fraction. The presence of these proteins in purified plasma membranes confirms the biochemical fractionation data demonstrating a protein-membrane interaction. Interestingly, a significant fraction of KIN1 and KIN2 proteins are present in purified cell wall preparations. Since protease protection experiments indicate that these proteins are confined to the cytoplasmic face of the plasma membrane and do not protrude into the periplasmic space, any association with the cell wall must be mediated through a protein complex.
Tibbetts
M
M
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459-0175.
Donovan
M
M
Roe
S
S
Stiltner
A M
AM
Hammond
C I
CI
D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002473:Cell Wall / Q000201:enzymology / Q000648:ultrastructure; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000032:analysis* / Q000235:genetics; D005800:Genes, Fungal; D005838:Genotype; D015151:Immunoblotting; D008565:Membrane Proteins; D010750:Phosphoproteins / Q000032:analysis* / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013104:Spheroplasts / Q000201:enzymology / Q000648:ultrastructure; D014461:Ultracentrifugation
D005656:Fungal Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins; C490209:KIN1 protein, S cerevisiae; C087572:KIN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases