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Vol. 16, Issue 2, 446-457, February 2005

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A Sorting Nexin PpAtg24 Regulates Vacuolar Membrane Dynamics during Pexophagy via Binding to Phosphatidylinositol-3-Phosphate

Yoshitaka Ano ^*, Takeshi Hattori ^*, Masahide Oku ^*, Hiroyuki Mukaiyama ^*, Misuzu Baba , Yoshinori Ohsumi , Nobuo Kato ^*, and Yasuyoshi Sakai ^*  

^* Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan; Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, Tokyo 112-8681, Japan; and Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan

Submitted September 27, 2004; Accepted November 5, 2004
Monitoring Editor: Howard Riezman

Diverse cellular processes such as autophagic protein degradation require phosphoinositide signaling in eukaryotic cells. In the methylotrophic yeast Pichia pastoris, peroxisomes can be selectively degraded via two types of pexophagic pathways, macropexophagy and micropexophagy. Both involve membrane fusion events at the vacuolar surface that are characterized by internalization of the boundary domain of the fusion complex, indicating that fusion occurs at the vertex. Here, we show that PpAtg24, a molecule with a phosphatidylinositol 3-phosphate-binding module (PX domain) that is indispensable for pexophagy, functions in membrane fusion at the vacuolar surface. CFP-tagged PpAtg24 localized to the vertex and boundary region of the pexophagosome-vacuole fusion complex during macropexophagy. Depletion of PpAtg24 resulted in the blockage of macropexophagy after pexophagosome formation and before the fusion stage. These and other results suggest that PpAtg24 is involved in the spatiotemporal regulation of membrane fusion at the vacuolar surface during pexophagy via binding to phosphatidylinositol 3-phosphate, rather than the previously suggested function in formation of the pexophagosome.

Abbreviations used: Snx(s), sorting nexin(s); PX, Phox homology; FYVE domain, Fab1-YOTB-Vac1-EEA1 homology domain; PtdIns, phosphoinoside; PtdIns(3)P, phosphatidylinositol 3-phosphate; PV-fusion, pexophagosome-vacuole fusion; MIPA, micropexophagy-specific membrane apparatus; PAS, preautophagosomal structure; GFP, green fluorescent protein; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; PTS1, peroxisome targeting signal type 1; GFP-PTS1, GFP fused to PTS1; FM4-64, N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide; GST, glutathione S-transferase; Aox, alcohol oxidase.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Corresponding author. E-mail address: ysakai{at}kais.kyoto-u.ac.jp.

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