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an alternative in-frame translational start site could initiate C3 expression within the cytosol (King et al., 2019 and unpublished data).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.