Cell-free reconstitution of microautophagic vacuole invagination and vesicle formation.

Details

Serval ID
serval:BIB_CEA08ED27D83
Type
Article: article from journal or magazin.
Collection
Publications
Title
Cell-free reconstitution of microautophagic vacuole invagination and vesicle formation.
Journal
Journal of Cell Biology
Author(s)
Sattler T., Mayer A.
ISSN
0021-9525 (Print)
ISSN-L
0021-9525
Publication state
Published
Issued date
2000
Peer-reviewed
Oui
Volume
151
Number
3
Pages
529-538
Language
english
Abstract
Many organelles change their shape in the course of the cell cycle or in response to environmental conditions. Lysosomes undergo drastic changes of shape during microautophagocytosis, which include the invagination of their boundary membrane and the subsequent scission of vesicles into the lumen of the organelle. The mechanism driving these structural changes is enigmatic. We have begun to analyze this process by reconstituting microautophagocytosis in a cell-free system. Isolated yeast vacuoles took up fluorescent dyes or reporter enzymes in a cytosol-, ATP-, and temperature-dependent fashion. During the uptake reaction, vacuolar membrane invaginations, called autophagic tubes, were observed. The reaction resulted in the transient formation of autophagic bodies in the vacuolar lumen, which were degraded upon prolonged incubation. Under starvation conditions, the system reproduced the induction of autophagocytosis and depended on specific gene products, which were identified in screens for mutants deficient in autophagocytosis. Microautophagic uptake depended on the activity of the vacuolar ATPase and was sensitive to GTPgammaS, indicating a requirement for GTPases and for the vacuolar membrane potential. However, microautophagocytosis was independent of known factors for vacuolar fusion and vesicular trafficking. Therefore, scission of the invaginated membrane must occur via a novel mechanism distinct from the homotypic fusion of vacuolar membranes.
Keywords
Adenosine Triphosphate/metabolism, Adenosine Triphosphate/pharmacology, Biological Transport/drug effects, Cell-Free System, Cytosol/drug effects, Cytosol/metabolism, Dextrans/metabolism, Endocytosis/drug effects, Fluorescein-5-isothiocyanate/analogs & derivatives, Fluorescein-5-isothiocyanate/metabolism, Fungal Proteins/metabolism, Genes, Fungal/genetics, Genes, Fungal/physiology, Guanosine 5'-O-(3-Thiotriphosphate)/metabolism, Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology, Hydrolysis/drug effects, Intracellular Membranes/drug effects, Intracellular Membranes/metabolism, Kinetics, Luciferases/metabolism, Membrane Fusion/drug effects, Microscopy, Fluorescence, Mutation/genetics, Phagocytosis/drug effects, Proton-Translocating ATPases/metabolism, Saccharomyces cerevisiae/cytology, Saccharomyces cerevisiae/drug effects, Temperature, Vacuolar Proton-Translocating ATPases, Vacuoles/chemistry, Vacuoles/drug effects
Pubmed
Web of science
Open Access
Yes
Create date
24/01/2008 15:06
Last modification date
20/08/2019 15:49
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