2 DISTINCT PATHWAYS FOR TARGETING PROTEINS FROM THE CYTOPLASM TO THE VACUOLE LYSOSOME/

Stress conditions lead to a variety of physiological responses at the cellular level. Autophagy is an essential process used by animal, plan t, and fungal cells that allows for both recycling of macromolecular c onstituents under conditions of nutrient limitation and remodeling the intracellular structure for cell differentiation. To elucidate the mo lecular basis of autophagic protein transport to the vacuole/lysosome, we have undertaken a morphological and biochemical analysis of this p athway in yeast. Using the vacuolar hydrolase aminopeptidase I (API) a s a marker, we provide evidence that the autophagic pathway overlaps w ith the biosynthetic pathway, cytoplasm to vacuole targeting (Cvt), us ed for API import. Before targeting, the precursor form of API is loca lized mostly in restricted regions of the cytosol as a complex with sp herical particles (termed Cvt complex). During vegetative growth, the Cvt complex is selectively wrapped by a membrane sac forming a double membrane-bound structure of similar to 150 nm diam, which then fuses w ith the vacuolar membrane. This process is topologically the same as m acroautophagy induced under starvation conditions in yeast (Baba, M., K. Takeshige, N. Baba, and Y. Ohsumi. 1994, J. Cell Biol. 124:903-913) . However, in contrast with autophagy, API import proceeds constitutiv ely in growing conditions. This is the first demonstration of the use of an autophagy-like mechanism for biosynthetic delivery of a vacuolar hydrolase. Another important finding is that when cells are subjected to starvation conditions, the Cvt complex is now taken up by an autop hagosome that is much larger and contains other cytosolic components; depending on environmental conditions, the cell uses an alternate path way to sequester the Cvt complex and selectively deliver API to the va cuole. Together these results indicate that two related but distinct a utophagy-like processes are involved in both biogenesis of vacuolar re sident proteins and sequestration of substrates to be degraded.