|Budget Amount *help
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1998: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1997: ¥1,900,000 (Direct Cost: ¥1,900,000)
We have investigated several mammalian proteins related to products of yeast genes essential for autophagy as candidates of molecules involved in mammalian autophagy, during the two-years research period.Obtained results are as follows.
1) LC3 : The rat LC3 is homologous to yeast Apg8p essential for autophagy. Using an antibody against LC3 and LC3 cDNA, we showed that about half of LC3 molecules were processed to 2-3 kDa-smaller form during 2-3 hours after synthesis and that the smaller form LC3 was specifically localized in the membrane organella involved in autophagy, autophagosomes, whereas the larger form LC3 was distributed over the cytoplasm.Starvation condition which induces autophagy and some drug treatment increase the smaller form LC3.These data suggest that the smaller form LC3 may play some role in mammalian autophagy.
2) SKD1 : The mouse SKD1 is an AAA-type ATPase highly homologous to the yeast Csclp implicated in autophagy.We generated a mutant SKD1 analogous to the dominant negative mutant Csclp. Overexpression of the mutant SKD1 in cultured animal cells caused formation of aberrant early endosomes and inhibition of recycling of transferrin receptor from early endosomes to the plasma membrane.From the results, we suggest that SKD1 regulates morphology and transport function of early endosomes, and then that such endosomal function may be required to proceed autophagy.
3) mAPG5 : We found that yeast Apg5p was conjugated covalently to Apgl2p and the conjugation was required for autophagy.Furthermore we also showed that their human homologue, hAPG5 and hAPG12, formed the covalent conjugation.To elucidate physiological meanings of autophagy at level of cell, tissue, and individual, we are generating mice deficient for the homologue of Apg5p, mAPG5.We already cloned genomic DNA of mAPG5 and will disrupt mAPG5 gene in ES cells.