Characterization of the Molecular Events of Autophagy

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DESCRIPTION: Eukaryotic cells adapt to environmental changes by altering their protein complements through synthesis and degradation. Cells that adapt poorly or improperly may either cease to exist (e.g., apoptosis) or become neoplastic (e.g., hepatoma). Cells adapt to low levels of amino acids by sequestering proteins and organelles for lysosomal degradation via a process called autophagy. The data from many laboratories suggest that autophagy is turned on during apoptosis and turned off during neoplastic growth implicating a role for autophagy in suppressing cancerous growth. Indeed, Beclin is a tumor suppressor that was originally shown to be required for autophagy in yeast. Our long-term goals are to characterize the molecular aspects of the regulation and mechanisms of cellular autophagy. We have characterized the degradation of peroxisomes and endogenous proteins by autophagy in the yeast Pichiapastoris under various environmental conditions. We have utilized this genetic model to identify 14 GSA genes that are required for glucose-induced selective autophagy. We have recently observed that during autophagy Gsa11 becomes associated with an organelle that is juxtaposed to the vacuole. In addition, this interaction requires the indirect or direct action of four additional GSA proteins including two protein kinases suggesting this event is highly regulated. Our results show that Gsa11 and its complex have a primary function in the sequestration of organelles for vacuole degradation. We propose that this organelle anchors at its surface a complex of proteins including Gsa11 that somehow organize the formation of the autophagic vacuole. In this application, we propose to examine the molecular and structural aspects of this organelle and the events required for the assembly of this complex in order to better understand its function. Our hypothesis is: Gsa11 must associate with a membrane-bound organelle prior to the sequestration events that occur during autophagy. We will utilize the versatility of our yeast model combined with a multidisciplinary approach of biochemical, cell biological, molecular biological, and genetic procedures to test our hypothesis. We will identify and characterize those GSA proteins that influence either directly or indirectly the formation of the Gsa11 complex. We will specifically evaluate the role of two protein kinases in the assembly of this complex. Finally, we will determine if the human homologue of Gsa11 is required for autophagy in a hepatoma cell line. The data obtained here will provide new insights into the events of sequestration of organelles for lysosomal degradation. In addition, with our new understanding of the molecular events of autophagy, we can begin to design clinical approaches by which to turn on autophagy and arrest neoplastic growth.