Membrane Protein Location-Dependent Regulation by PI3K (III) and Rabenosyn-5 in Drosophila Wing Cells Masato Abe1¤a, Yuka Setoguchi1, Tsubasa Tanaka3¤b, Wakae Awano 2 , Kuniaki Takahashi4, Ryu Ueda4, Akira Nakamura3, Satoshi Goto1,5* 1 Glycobiology and Glycotechnology Research Group, Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan, 2 Mutant Flies Laboratory, Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan, 3 Laboratory for Germline Development, RIKEN Center for Development Biology, Hyogo, Japan, 4 Invertebrate Genetics Laboratory, National Institute of Genetics, Shizuoka, Japan, 5 Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Saitama, Japan Abstract The class III phosphatidylinositol-3 kinase (PI3K (III)) regulates intracellular vesicular transport at multiple steps through the production of phosphatidylinositol-3-phosphate (PI(3)P). While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development. In this study, we examined in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) in Drosophila wing primordial cells, which are polarized along the apical-basal axis. Knockdown of the PI3K (III) subunit Vps15 resulted in an accumulation of the apical junctional proteins DE-cadherin and Flamingo and also the basal membrane protein b-integrin in intracellular vesicles. By contrast, knockdown of PI3K (III) increased lateral membrane- localized Fasciclin III (Fas III). Importantly, loss-of-function mutation of Rbsn-5 recapitulated the aberrant localization phenotypes of b-integrin and Fas III, but not those of DE-cadherin and Flamingo. These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes. Citation: Abe M, Setoguchi Y, Tanaka T, Awano W, Takahashi K, et al. (2009) Membrane Protein Location-Dependent Regulation by PI3K (III) and Rabenosyn-5 in Drosophila Wing Cells. PLoS ONE 4(10): e7306. doi:10.1371/journal.pone.0007306 Editor: Andreas Bergmann, University of Texas MD Anderson Cancer Center, United States of America Received March 25, 2009; Accepted September 11, 2009; Published October 2, 2009 Copyright: 2009 Abe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Ministry of Education, Culture Sports, Science, and Technology of Japan and the Japan Science and Technology Agency (to S.G.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sgoto@mitils.jp ¤a Current address: Biological Products Department, Takeda Pharmaceutical Company Limited, Yamaguchi, Japan ¤b Current address: Department of Cell & Tissue Biology, University of California San Francisco, San Francisco, California, United States of America Introduction Cell polarity along the apical-basal axis is essential for the function of epithelial cells. This polarity is formed and maintained by distinct localization of membrane spanning and associated proteins, to apical, lateral or basal membrane domains. Membrane proteins localized to the apical or basolateral plasma membrane are endocytosed into early and apical or basolateral endosomes. For example, horseradish peroxidase (HRP) administered to the apical cell surface is incorporated into the apical early endosome. By contrast, HRP or dimeric IgA administered to the basolateral cell surface or transferring receptor (TfR) in the basolateral domain are internalized into the basolateral early endosome, which remain distinct [1–4]. Sorting of proteins for transcytosis, recycling and degradation takes place in these early endosomes. The proteins, incorporated into apical and basolateral early endosomes, meet in common endosomes, a process that can be observed within 15 min after the onset of internalization in MDCK cells [4]. The significance of keeping the apical and basolateral early endosomes distinct is thought to ensure that proteins from the apical and basolateral plasma membrane remain apart before the sorting processes proceeds. Although it is plausible that the trafficking of proteins in distinct membrane domains is regulated differently, the factors involved in such a differential regulation remain elusive. One of the key molecules regulating membrane trafficking is PI3K (III), a heterodimer of Vps34p and Vps15p/p150, which produces phosphatidylinositol-3-phosphate (PI(3)P) [5–8]. PI(3)P is found to localize with early endosome and internal vesicles of multivesicular bodies (MVBs) in mammalian cells in culture [9]. Genetic and pharmacological analysis, using yeast and mamma- lian cells in culture, suggests that PI3K (III) is required for five distinct processes. These are: (i) the fusion of clathrin-coated vesicles and early endosomes as well as the fusion between early endosomes [10–13]; (ii) the recycling from early endosomes back to the Golgi complex or other destinations [14,15]; (iii) the entry of proteins into the lysosomal degradation pathway [16–22]; (iv) the formation of internal vesicles of MVBs [23,24] and (v) autophagy [25,26]. Moreover, inactivation of PI3K (III) by Vps34 mutation leads to an expansion of the outer nuclear membrane and an abnormal reduction of the LDL receptor at the apical membrane in C. elegans [27]. In Drosophila, dVps34 mutation results in defective endocytosis of the apical membrane protein Notch and a defective onset of autophagy [28]. It has been suggested that PI3K (III) utilizes different effectors at apical and basolateral endosomes [15]. However, the role of PI3K (III) in the regulation of protein localization at different membrane domains has remained unclear. To understand the various functions of PI3K (III), it is crucial to clarify which downstream effectors are involved in each of the PLoS ONE | www.plosone.org 1 October 2009 | Volume 4 | Issue 10 | e7306