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dictyNews Volume 43 Number 09
dictyNews
Electronic Edition
Volume 43, number 9
May 5, 2017
Please submit abstracts of your papers as soon as they have been
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http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit.
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Abstracts
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The two Dictyostelium discoideum autophagy 8 proteins have distinct
autophagic functions
Susanne Meßling, Jan Matthias, Qiuhong Xiong, Sarah Fischer and
Ludwig Eichinger
Center for Biochemistry, Institute of Biochemistry I, Medical Faculty,
University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Cologne, Germany
European Journal of Cell Biology, in press.
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.
submitted by: Ludwig Eichinger [ludwig.eichinger@uni-koeln.de]
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[End dictyNews, volume 43, number 9]
