PARENT SESSION

(M391) CHARACTERIZATION OF INTERACTION BETWEEN THE AUTOPHAGY PROTEIN BECLIN AND GAMETOGENETIN: IMPLICATIONS FOR MALE GERM CELL SURVIVAL.

Welbern, Vanessa¹, Alinea, Corinne¹, Rucker, Edmund¹, ¹ University of Missouri-Columbia, Columbia, MO

ABSTRACT- Programmed cell death (PCD) is a genetic program through which cells are eliminated during embryonic and adult stages of development. Apoptosis, or PCD type I, is predominantly a caspase-dependent process mediated through the mitochondria; autophagy, or PCD type II, involves recycling of proteins and organelles through lysosomes. We are interested in determining what apoptosis-related proteins are needed for the maintenance of male germ cells and sustained fertility. Altered levels of apoptosis are known to give rise to pathophysiological conditions and disease. Recently, autophagy has gained a more prominent role due to: 1) association with disease progression (e.g. neurodegenerative disorders, cancer), and 2) biochemical links to apoptotic proteins. With respect to the latter, we used the anti-apoptotic protein Bcl-xL as a bait and identified beclin in a high stringency yeast-two hybrid screen using a testis cDNA library of 1 x 106 independent clones. Immunohistochemistry and co-immunoprecipitation revealed that Bcl-xL and beclin interact and co-localize within the testis. Beclin is an autophagy protein required for the formation of autophagosomes from the Golgi complex and is a known tumor suppressor gene. To gain a more complete understanding how these proteins might regulate germ cell survival, we performed a second yeast two-hybrid screen using beclin as the bait protein and obtained 113 clones from a high stringency screen. Sequence analysis of 6 of these clones revealed the insert to be encoding for the protein gametogenetin, which has been causally linked to the phenotype of the jsd mutant mouse. To characterize the domains involved in the interaction between beclin and gametogenetin, we have prepared a series of GST-fusion proteins for in vitro co-immunoprecipitation assays. In addition, we are profiling the expression of beclin in the prepubetal and adult testis by RT-PCR and immunohistochemistry. Through these studies we hope to understand the regulation of germ cell attrition by apoptosis and autophagy.

KEY WORDS: yeast two-hybrid, beclin, gametogenetin, autophagy