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First published online July 1, 2009
doi: 10.1242/10.1242/jcs.046391

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Comparative analysis of ESCRT-I, ESCRT-II and ESCRT-III function in Drosophila by efficient isolation of ESCRT mutants

Thomas Vaccari^1,*,, Tor Erik Rusten², Laurent Menut¹, Ioannis P. Nezis², Andreas Brech², Harald Stenmark² and David Bilder^1,

¹ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94702, USA
² Centre for Cancer Biomedicine and Department of Biochemistry, the Norwegian Radium Hospital, University of Oslo, Montebello, N-0310 Oslo, Norway

Authors for correspondence (e-mails: thomas.vaccari{at}ifom-ieo-campus.it; bilder{at}berkeley.edu)

Accepted 16 April 2009

ESCRT proteins were initially isolated in yeast as a single functional set of conserved components controlling endosomal cargo sorting and multivesicular body (MVB) biogenesis. Recent work has suggested that metazoan ESCRT proteins might have more functionally diverse roles, but the limited availability of ESCRT mutants in species other than yeast has hampered a thorough analysis. Here, we used a genetic screening strategy based on both cell-autonomous and non-autonomous growth-promotion phenotypes to isolate null mutations in nearly half of the ESCRT-encoding genes of Drosophila, including components of ESCRT-I, ESCRT-II and ESCRT-III complexes. All ESCRT components are required for trafficking of ubiquitylated proteins and are required to prevent excess Notch and EGFR signaling. However, cells lacking certain ESCRT-III components accumulate fewer ubiquitylated molecules in endosomes and display reduced degrees of cell proliferation compared with those lacking components of ESCRT-I and ESCRT-II. Moreover, although we find by ultrastructural analysis that MVB formation is impaired in ESCRT-I and ESCRT-II mutant cells, MVB biogenesis still occurs to some degree in ESCRT-III mutant cells. This work highlights the multiple cell biological and developmental roles of ESCRT proteins in Drosophila, suggests that the metazoan ESCRT-I, ESCRT-II and ESCRT-III complexes do not serve identical functions, and provides the basis for an extensive analysis of metazoan ESCRT function.

Key words: ESCRT, MVB sorting, Endocytosis, Drosophila, Tumor suppression genes

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