ATG9 is a membrane protein that is essential for autophagy and is considered to be directly involved in the early steps of autophagosome formation. Yeast Atg9 is mainly localized to small vesicles (Atg9 vesicles), whereas mammalian ATG9A is reportedly localized to the trans-Golgi network, the endosomal compartment, and other unidentified membrane structures. To dissect the ATG9A-containing membranes, we examined the subcellular localization of ATG9A and performed immunoisolation of those membranes. ATG9A-green fluorescent protein in human culture cells was observed as numerous puncta that move rapidly throughout the cytoplasm. We isolated these cytoplasmic membranes and found that they were small vesicles that resemble the yeast Atg9 vesicle. One of the proteins obtained via proteomic analyses of the mammalian ATG9A vesicle was Rab1, a small GTPase that is essential in endoplasmic reticulum-to-Golgi vesicle trafficking. Knockdown studies of Rab1B showed a suppression of autophagy. In these Rab1B-depleted cells, ATG9A accumulated in intermediate membrane structures at autophagosome formation sites. These results indicate that Rab1B is involved in regulating the proper development of autophagosomes.-Kakuta, S., Yamaguchi, J., Suzuki, C., Sasaki, M., Kazuno, S., Uchiyama, Y. Small GTPase Rab1B is associated with ATG9A vesicles and regulates autophagosome formation.
Keywords: autophagy; electron microscopy; mass spectrometry.
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