Targeting and modification of Yck2 in Saccharomyces cerevisiae.

摘要

Casein kinase I (CK1) enzymes comprise a large subfamily of acidic Ser/Thrspecific protein kinases that is strongly conserved from yeast to humans. CK1s are involved in numerous signaling pathways and cellular processes. Aberrant CK1 activity has been associated with Parkinson's disease, Down syndrome, and Alzheimer's disease. While the ubiquitous distribution of numerous CK1s in higher eukaryotes makes their study exceedingly complex, the budding yeast Saccharomyces cerevisiae encodes only four CK1 enzymes.; The Yck1 and Yck2 (y&barbelow;east c&barbelow;asein k&barbelow;inase I homologue) proteins are plasma membrane (PM)-associated CK1 isoforms required for bud morphogenesis, cytokinesis, endocytosis, and other cellular processes. Yckp PM localization is critical for function, as soluble mutant protein provide insufficient biological activity to sustain growth. PM targeting occurs through association with vesicular intermediates of the classical secretory pathway, yet Yck1p and Yck2p lack predicted signal sequences or obvious transmembrane domains. Both kinases possess palmitoylated carboxyl-terminal -Cys-Cys sequences, but the mechanisms behind PM localization of palmitoylated proteins with no other lipid modification are poorly understood.; The following results show that Yck1p lacking its carboxyl-terminal Cys residues fails to associate with membranes. Substitution of one of these residues with a signal for farnesylation allows secretory pathway-mediated PM targeting and biological function, suggesting that hydrophobic modification is required for interaction with secretory membranes. Deletion analysis indicates that in addition to the -Cys-Cys signal, the final 28 residues are critical for membrane association and PM targeting.; The Yck2p variant carrying the farnesyl transferase signal targets normally to the plasma membrane, but no longer requires Akr1p, the palmitoyl-transferase for Yck2p, for membrane association. Carboxyl-terminal deletions of sequences believed to affect Yck2p membrane association were created in this variant. All of the sequences identified as important for PM association are required only for Akr1p-dependent modification. Furthermore, palmitoylation is sufficient for specific association with secretory vesicles destined for the PM, and residues 499--546 are sufficient for minimal Yck2p palmitoylation and PM localization. Both carboxyl-terminal Cys residues are palmitoylated, and dual acylation is required for efficient membrane association. Finally, a model is presented that could apply generally to the targeting of palmitoylated proteins.