Involvement of intracellular C3 in autophagy
Our study therefore identified C3 as a relevant effector in
cytoprotective autophagy, but also revealed possible derivation of C3
endogenously expressed within the cytosol. C3 is known primarily as a
secreted protein, raising questions as to how it may interact with
cytosolic ATG16L1 in the context of normal homeostasis. We found that an
alternative in-frame translational start site could initiate C3
expression within the cytosol (King et al., 2019 and unpublished data).
The C3 mRNA contains in-frame AUG codons immediately downstream of the
encoded signal peptide, the utilization of which produces non-secreted,
cytosolic C3. Indeed, site-directed mutagenesis of the first AUG codon
of the C3 coding sequence did not affect translation of a non-secreted,
cytosolic form C3 that was not associated with organelle/membrane
structures, as opposed to wild type C3 that is found abundantly in
organelle/membrane fractions, which include the ER and golgi components
of the secretory pathway. The ’scanning’ model of translational
initiation, in which the translation initiation complex uses the first
available AUG codon (Kozak, 1978), could explain aberrant use of a
non-physiological start site when the canonical site is removed, but
cytosolic C3 was also expressed from the endogenous gene with an intact
canonical AUG start site. Introduction of a frame shift within the
signal peptide-coding sequence of C3 by genomic CRISPR/Cas9 gene editing
of C3-expressing cells, without altering the canonical start codon,
completely prevented expression of secreted C3, but did not affect
detection of cytosolic C3 (King et al., 2019), supporting constituitive
use of additional downstream translational start sites in C3-expressing
cells.
Exogenous addition of C3 to cell culture medium did not rescue the
inhibited autophagy phenotype in C3 knockout cells (King et al., 2019),
emphasizing the importance of intrinsic intracellular C3 to preserve
optimal autophagic function. However, it can not yet be excluded that
extracellular C3 might be recruited to damaged organelles for
utilization in the autophagy pathway, in a mechanism similar to
C3-targeted xenophagy. C3 can be internalized from the extracellular
space (Elvington, Liszewski, Bertram, Kulkarni, & Atkinson, 2017;
Kremlitzka et al., 2019), although a mechanism has not been demonstrated
by which C3 within the lumen of internalised membrane-bound compartments
should cross into the cytosol to access the autophagy machinery.