Recycling of Vps68 depends on retromer and Mvp1/SNX8.

Here, we analyzed the recycling of the Vps55/Vps68 complex in the yeast endocytic pathway. The two proteins seem to form a functional unit. Single deletions of VPS55 or VPS68 affected the turnover of the endocytic cargo protein Ste6 to the same degree. The double deletion had no additive effect on Ste6 turnover. Vps55 and Vps68 were dependent on each other for proper cellular localization. Under normal conditions, the sfGFP-tagged proteins localized to punctate structures; but, when the corresponding partner protein was missing, staining of the vacuolar lumen was observed. This suggests that the orphaned proteins are degraded in the vacuole via the multivesicular bodies (MVB) pathway. A tyrosine-based recycling signal was identified in the cytosolic tail of Vps68. This signal completely restored retromer-dependent recycling and function of a Vps10 variant devoid of its own recycling signals. In line with this finding, Vps68 recycling turned out to be dependent on both retromer and Mvp1/SNX8. This finding was unexpected, since Vps55 recycling seems to depend solely on Mvp1. In co-immunoprecipitation experiments, a weak co-immunoprecipitation signal was detected between Mvp1 and the retromer subunit Vps26, indicating a physical association between Mvp1 and retromer.