Miki Tsukada

Autophagy in the yeast is similar to that in mammalian cells. A mutant designated as apg1 (autophagy) defective in accumulation of autophagic bodies in the vacuoles was isolated by selection using a light microscope from a mutagenized proteinase-deficient strain. In the apg1 strain, which has normal vacuolar proteinases, nitrogen starvation did not induce protein degradation. The apg1 mutant lost its viability faster than wild-type cells during nitrogen starvation. By using the loss of viability as a first screening test, 75 other apg mutants were selected. These apg mutants including apg1 fell into 15 complementation groups. Genetic analyses of representative apg mutants revealed that they all had single recessive chromosomal mutations. Strains with each apg mutation were defective in protein degradation in the vacuoles induced by nitrogen starvation and homozygous diploids for each apg mutation did not sporulate. These results on the apg mutants suggest that autophagy via autophagic bodies is indispensable for protein degradation in the vacuoles under starvation conditions, and that at least 15 APG genes are involved in autophagy in yeast.

In Saccharomyces cerevisiae, the YPT6 gene encodes the homologue of the mammalian Rab6 protein found in the Golgi apparatus. Deletion of YPT6 in yeast produces a phenotype showing temperature-sensitive growth and partial missorting of the vacuolar enzyme, carboxypeptidase Y. To identify proteins that might: (1) interact with Ypt6p; or (2) act in the same pathway, we have isolated four multicopy suppressors, named SYS1, SYS2, SYS3 and SYS5, that can complement the temperature-sensitive growth phenotype of the ypt6 null mutant. On high expression, these genes are also able to partially suppress the missorting of carboxypeptidase Y.SYS2 on a multicopy plasmid suppresses in addition the temperature-sensitive phenotype of sec7-1, a mutant defective in transport between and from the Golgi compartment. Gene disruption of SYS1 and SYS2 did not result in significant growth defects. However, deletion of SYS1 and/or SYS2 in the ypt6 null mutant enhances defects in vacuolar protein sorting and in cell growth. Whereas protein secretion was not significantly affected in these mutants, the processing of alpha-factor precursor by the Kex2 protease was inhibited, suggesting a function of YPT6 and its null mutant suppressors in transport between the late Golgi and a prevacuolar, endosome-like compartment.