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J. Biol. Chem., Vol. 279, Issue 11, 9987-9996, March 12, 2004

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Determination of Four Sequential Stages during Microautophagy in Vitro^*

Joachim B. Kunz, Heinz Schwarz¶, and Andreas Mayer||**

From the Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstrasse 37-39 and ^¶Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35, 72076 Tübingen, Germany

Microautophagy is the transfer of cytosolic components into the lysosome by direct invagination of the lysosomal membrane and subsequent budding of vesicles into the lysosomal lumen. This process is topologically equivalent to membrane invagination during multivesicular body formation and to the budding of enveloped viruses. Vacuoles are lysosomal compartments of yeasts. Vacuolar membrane invagination can be reconstituted in vitro with purified yeast vacuoles, serving as a model system for budding of vesicles into the lumen of an organelle. Using this in vitro system, we defined different reaction states. We identified inhibitors of microautophagy in vitro and used them as tools for kinetic analysis. This allowed us to characterize four biochemically distinguishable steps of the reaction. We propose that these correspond to sequential stages of vacuole invagination and vesicle scission. Formation of vacuolar invaginations was slow and temperature-dependent, whereas the final scission of the vesicle from a preformed invagination was fast and proceeded even on ice. Our observations suggest that the formation of invaginations rather than the scission of vesicles is the rate-limiting step of the overall reaction.

Received for publication, July 21, 2003 , and in revised form, December 12, 2003.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 446 (to A. M.) and Studienstiftung des Deutschen Volkes (to J. B. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Universitäts Kinderklinik III, Abt. für Pädiatrische Onkologie, Hämatologie und Immunologie, Im Neuenheimer Feld 153, 69120 Heidelberg.

^|| Present address: Département de Biochimie, Université de Lausanne, Chemin des Boveresses 155, 1066 Epalinges, Switzerland.

^** To whom correspondence should be addressed. Tel.: 49-7071-601850; Fax: 49-7071-601455; E-mail: andreas.mayer{at}tuebingen.mpg.de.

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