A ROLE FOR CLATHRIN IN THE SORTING OF VACUOLAR PROTEINS IN THE GOLGI-COMPLEX OF YEAST

We have investigated the role of clathrin in vacuolar protein sorting using yeast strains harboring a temperature-sensitive allele of clathrin heavy chain (chc1-ts). After a 5 min incubation at the non-permissive temperature (37-degrees-C), the chc1-ts strains displayed a severe defect in the sorting of lumenal vacuolar proteins. Sorting of a vacuolar membrane protein, alkaline phosphatase, and transport to the surface of a cell wall protein, was not affected at 37-degrees-C. In chc1-ts cells incubated at 37-degrees-C, secretion of the missorted lumenal vacuolar protein carboxypeptidase Y (CPY) was blocked by the sec1 mutation which prevents fusion of secretory vesicles to the plasma membrane. Unexpectedly, chc1-ts cells incubated for extended periods at 37-degrees-C regained the ability to sort CPY. Cells carrying deletions of the CHC1 gene (chc1-DELTA) also sorted CPY to the vacuole even when subjected to temperature shifts. Vacuolar delivery of CPY in chc1-DELTA cells was not blocked by sec1 suggesting that transport does not occur by secretion and endocytosis. These results provide in vivo evidence that clathrin plays a role in the Golgi complex in sorting of vacuolar proteins from the secretory pathway. With time, however, yeast cells lacking functional clathrin heavy chains are able to adapt in a way that allows restoration of vacuolar protein sorting in the Golgi complex. These conclusions clarify previous studies of chc1-DELTA cells which raised the possibility that clathrin is not involved in vacuolar protein sorting.