Akr1p-dependent palmitoylation of the Yck2p C-terminus is sufficient for Sec-dependent plasma membrane targeting.
Praveen Babu, Lucy C. Robinson
Biochemistry and Mol. Biology, LSU Health Sciences Center, 1501 Kings Highway, Shreveport, LA, 71130, USA
The Yck2 protein kinase is a plasma membrane (PM)-associated casein kinase 1 that is palmitoylated on one or both of its two C-terminal Cys residues. We have shown that Yck2p traffics to the PM on secretory vesicles. Akr1p, the palmitoyl transferase for Yck2p, is located on Golgi membranes, suggesting that Yck2p first associates with Golgi membranes, then is recruited to budding PM-bound vesicles. We show here that the final 48 C-terminal residues are necessary and sufficient for Yck2p PM targeting. We previously described normal PM targeting of a Yck2p variant with the final 5 residues of Ras2p substituting for the two Yck2p terminal Cys residues. This variant no longer requires Akr1p for membrane association, but targets normally. C-terminal deletions that affect Yck2p PM association were made in the context of this variant Yck2p. All of the sequences previously shown to be important for PM association are required only for Akr-dependent modification. Further, palmitoylation efficiency in vivo correlates with PM localization, and both Cys residues must be palmitoylated for optimal membrane association. Thus, palmitoylation of both terminal Cys residues appears sufficient for specific association of Yck2p with secretory vesicles destined for the PM. We are continuing mutagenesis of C-terminal residues to assess Akr1p-Yck2p interaction requirements, and testing whether Sec-dependent Yck2p targeting is accomplished by association with membrane microdomains at the Golgi.
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