Ric1p and the Ypt6p GTPase function in a common pathway required for localization of trans-Golgi network membrane proteins

In Saccharomyces cerevisiae, clathrin is necessary for localization of tran s-Golgi network (TGN) membrane proteins, a process that involves cycling of TGN proteins between the TGN and endosomes. To characterize further TGN pr otein localization, we applied a screen for mutations that cause severe gro wth defects in combination with a temperature-sensitive clathrin heavy chai n. This screen yielded a mutant allele of RIC1. Cells carrying a deletion o f RIC1 (ric1 Delta) mislocalize TGN membrane proteins Kex2p and Vps10p to t he vacuole. Delivery to the vacuole occurs in ric1 Delta cells also harbori ng end3 Delta to block endocytosis, indicative of a defect in retrieval to the TGN rather than sorting to endosomes. SYS1, originally discovered as a multicopy suppressor of defects caused by the absence of the Rab GTPase YPT 6, was identified as a multicopy suppressor of ric1 Delta. Further comparis on of ric1 Delta and ypt6 Delta cells demonstrated identical phenotypes. Mu lticopy plasmids expressing v-SNAREs Gos1p or Ykt6p, but not other v- and t -SNAREs, partially suppressed phenotypes of ric1 Delta and ypt6 Delta cells . SLY1-20, a dominant activator of the cis-Golgi network t-SNARE Sed5p, als o functioned as a multicopy suppressor. Because Gos1p and Ykt6p interact wi th Sed5p, these results raise the possibility that TGN membrane protein loc alization requires Ric1p- and Ypt6p-dependent retrieval to the cis-Golgi ne twork.