Universität zu Köln

Messling, Susanne, Matthias, Jan, Xiong, Qiuhong, Fischer, Sarah ORCID: 0000-0001-6227-3950 and Eichinger, Ludwig ORCID: 0000-0003-1594-6117 (2017). The two Dictyostelium discoideum autophagy 8 proteins have distinct autophagic functions. Eur. J. Cell Biol., 96 (4). S. 312 - 325. JENA: ELSEVIER GMBH, URBAN & FISCHER VERLAG. ISSN 1618-1298

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Abstract

Autophagy is a highly conserved cellular degradation pathway which is crucial for various cellular processes. The autophagic process is subdivided in the initiation, autophagosome maturation and lysosomal degradation phases and involves more than forty core and accessory autophagy-related (ATG) proteins. Autophagy 8 (ATG8, in mammals LC3) is a well-established marker of autophagy and is linked to the autophagic membrane from initiation until fusion with the lysosome. We generated single and double knock-out mutants of the two Dictyostelium paralogues, ATG8a and ATG8b, as well as strains that expressed RFP-ATG8a and/or GFP-ATG8b, RFP-ATG8b, RFP-GFP-ATG8a or RFP-GFP-ATG8b in different knock-out mutants. The ATG8b(-) mutant displayed only subtle phenotypic changes in comparison to AX2 wild-type cells. In contrast, deletion of ATG8a resulted in a complex phenotype with delayed development, reduced growth, phagocytosis and cell viability, an increase in ubiquitinylated proteins and a concomitant decrease in proteasomal activity. The phenotype of the ATG8a(-)/b(-) strain was, except for cell viability, in all aforementioned aspects more severe, showing that both proteins function in parallel during most analysed cellular processes. Immunofluorescence analysis of knock-out strains expressing either RFP-GFP-ATG8a or RFP-GFP-ATG8b suggests a crucial function for ATG8b in autophagosome-lysosome fusion. Quantitative analysis of strains expressing RFP-ATG8a, RFP-ATG8b, or RFP-ATG8a and GFP-ATG8b revealed that ATG8b generally localised to small and large vesicles, whereas ATG8a preferentially co-localised with ATG8b on large vesicles, indicating that ATG8b associated with nascent autophagosomes before ATG8a, which is supported by previous results (Matthias et al., 2016). Deconvoluted confocal fluorescence images showed that ATG8b localised around ATG8a and was presumably mainly present on the outer membrane of the autophagosome while ATG8a appears to be mainly associated with the inner membrane. In summary, our data show that ATG8a and ATG8b have distinct functions and are involved in canonical as well as non-canonical autophagy. The data further suggest that ATG8b predominantly acts as adapter for the autophagy machinery at the outer and ATG8a as cargo receptor at the inner membrane of the autophagosome. (C) 2017 The Authors. Published by Elsevier GmbH.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Messling, Susanne UNSPECIFIED UNSPECIFIED UNSPECIFIED Matthias, Jan UNSPECIFIED UNSPECIFIED UNSPECIFIED Xiong, Qiuhong UNSPECIFIED UNSPECIFIED UNSPECIFIED Fischer, Sarah UNSPECIFIED orcid.org/0000-0001-6227-3950 UNSPECIFIED Eichinger, Ludwig UNSPECIFIED orcid.org/0000-0003-1594-6117 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-228622
DOI:	10.1016/j.ejcb.2017.03.014
Journal or Publication Title:	Eur. J. Cell Biol.
Volume:	96
Number:	4
Page Range:	S. 312 - 325
Date:	2017
Publisher:	ELSEVIER GMBH, URBAN & FISCHER VERLAG
Place of Publication:	JENA
ISSN:	1618-1298
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language UBIQUITIN-PROTEASOME SYSTEM; SWISS-MODEL; SELECTIVE AUTOPHAGY; MONOCLONAL-ANTIBODIES; REGULATE AUTOPHAGY; CELLS; DISEASE; BIOGENESIS; MEMBRANE; ATG8 Multiple languages Cell Biology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/22862