Role of Hrs in maturation of autophagosomes in mammalian cells

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Abstract

Autophagy is an evolutionarily conserved system responsible for the degradation of cellular components and contributes to the increasing of amino acid pool, organelle turnover, and elimination of intracellular bacteria. The molecular process of autophagy is still unclear. Here we demonstrate that Hrs, a master regulator in endosomal protein sorting, plays critical roles for the autophagic degradation of non-specific proteins and Streptococcus pyogenes. We found that Hrs containing FYVE domain is localized to autophagosomes. Hrs depletion resulted in a significant decrease in the number of mature autophagosomes (autophagolysosomes) detected by the co-localization of autophagosome marker LC3 and lysosome marker LAMP-1. In contrast, formation of the primary autophagosome, detected by LC3 immunoblotting and lysosomal degradation of non-specific proteins, were not significantly altered by Hrs depletion. Based on these results, we propose a novel function of Hrs, as a crucial player in the maturation of autophagosomes.

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Materials and methods

Cells. HeLa cells (American Type Culture Collection), Hrs-deficient mouse embryonic fibroblasts (HRSd), and an HRSd subline stably transfected with the hrs gene (HRSw) [14] were maintained in DMEM containing 10% FCS and antibiotics. A HeLa subline stably transfected with LC3-EGFP (GFP-LC3-HeLa) was previously described [4]. For amino acid starvation, cells were first extensively washed with Hanks’ balanced salt solution (HBSS) and further incubated in HBSS for the indicated time periods.

RNA

Localization of Hrs to LC3-positive autophagosomes mediated by its FYVE domain

To elucidate the involvement of Hrs in cellular autophagy, we first examined whether Hrs has any direct association with autophagosomes, using the microtubule-associated protein (MAP) light chain 3 (LC3) fused with green fluorescent protein (GFP). In mammals, GFP-tagged LC3 is used as a marker for intracellular autophagy. In HeLa cells stably transfected with LC3-GFP (GFP-LC3-HeLa) and grown in regular culture medium, LC3 localized throughout the cytoplasm with no significant puncta, while Hrs

Discussion

This study reveals a potential molecular mechanism to better understand the autophagy machinery. In mammalian cells, the maturation of autophagosomes requires fusion with endocytic compartments, including lysosomes. A recent report suggests that endosomes may provide autophagosomes with one or more factors required for their fusion with lysosomes [17]. In cells overexpressing SKD1E235Q, numerous autophagosomes and few autophagolysosomes are observed, which results in the inhibition of

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, a Grant-in-Aid for Scientific Research in Priority Areas, from the Ministry of Education, Science, Sports and Culture in Japan, and a Grant-in-Aid from Hiromi Medical Research Foundation. K.T. is a JSPS Fellow. We thank L. Aayam and L.C. Ndhlovu for critical reading of the manuscript.

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