Determination of four sequential stages during microautophagy in vitro.


Autoria(s): Kunz J.B.; Schwarz H.; Mayer A.
Data(s)

2004

Resumo

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.

Identificador

http://serval.unil.ch/?id=serval:BIB_B6854FC4ACBA

isbn:0021-9258 (Print)

pmid:14679207

doi:10.1074/jbc.M307905200

isiid:000220050400042

Idioma(s)

en

Fonte

Journal of Biological Chemistry, vol. 279, no. 11, pp. 9987-9996

Palavras-Chave #Autophagy; Cell-Free System; Colchicine/pharmacology; Cytosol/metabolism; Dose-Response Relationship, Drug; Inhibitory Concentration 50; Intracellular Membranes/metabolism; Kinetics; Luciferases/metabolism; Lysosomes/metabolism; Microscopy, Electron; Nocodazole/pharmacology; Saccharomyces cerevisiae/metabolism; Temperature; Time Factors; Vacuoles/metabolism
Tipo

info:eu-repo/semantics/article

article