Young, A.R.J.; (2007) Characterisation of mammalian Atg9 and its role in autophagy. Doctoral thesis , University of London.

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Abstract

Cells undergo autophagy or self-eating as a means of recycling their constituents in order to maintain homeostasis. Autophagy is up regulated by stress, including amino acid deprivation for which it is best characterised. Upon amino acid starvation double or multiple lamellar vesicles termed autophagic vacuoles (AV) or autophagosomes appear throughout the cell's cytoplasm. From their content they can be seen to have sequestered cytoplasm, often including organelles. Screens for autophagy defective mutants in Saccharomyces cerevisiae resulted in the AuTophaGy (ATG) genes. I have studied the ubiquitously expressed mammalian orthologue of Atg9p (Atg9Ll), a multi-spanning transmembrane protein shown to be essential in yeast for autophagy. I studied Atg9Ll in the hope that, as it is a multi-spanning transmembrane protein, it might provide clues as to the origin of the autophagosomal membranes. Initially addressing the protein's topology I show that both the N-and C-termini of Atg9L1 are cytosolic, and predict that Atg9Ll spans the membrane six times. siRNA mediated depletion of Atg9L 1 using adenovirus in hepatocytes did not significantly reduce the number of early or initial autophagosomes (AVi) arising upon starvation but did result in smaller AVi and a greater number of degradative autophagosomes (AVd). By immunofluorescence and subcellular fractionation analysis I found that Atg9Ll is located in the TGN and late endosomes. In immunofluorescence Atg9Ll colocalises with TGN46, the cation-independent mannose-6-phosphate receptor (CI-MPR), Rab7 and Rab9. Amino acid starvation alters the distribution of Atg9L1 causing a relocalisation of the protein from the TGN to a peripheral, endosomal, population occasionally colocalising with GFP-LC3, a well characterised marker of autophagosomes. siRNA mediated depletion of the mammalian homologue of Atglp, ULK1, inhibits the starvation dependent relocalisation of Atg9Ll. The starvation induced relocalisation of Atg9Ll requires PtdIns-3-kinase activity, and is reversed after restoration of amino acids. I speculate that starvation induced autophagy may rely on an alteration of the steady state trafficking of Atg9Ll, in a ULK1 dependent manner.

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