Abstract
Selective autophagy is mediated by cargo receptors that link the cargo to the isolation membrane via interactions with Atg8 proteins. Atg8 proteins are localized to the membrane in an ubiquitin-like conjugation reaction, but how this conjugation is coupled to the presence of the cargo is unclear. Here we show that the S. cerevisiae Atg19, Atg34 and the human p62, Optineurin and NDP52 cargo receptors interact with the E3-like enzyme Atg12~Atg5-Atg16, which stimulates Atg8 conjugation. The interaction of Atg19 with the Atg12~Atg5-Atg16 complex is mediated by its Atg8-interacting motifs (AIMs). We identify the AIM-binding sites in the Atg5 subunit and mutation of these sites impairs selective autophagy. In a reconstituted system the recruitment of the E3 to the prApe1 cargo is sufficient to drive accumulation of conjugated Atg8 at the cargo. The interaction of the Atg12~Atg5-Atg16 complex and Atg8 with Atg19 is mutually exclusive, which may confer directionality to the system.
Highlights
Macro-autophagy is a conserved pathway for the delivery of cytoplasmic material into the lysosomal system for degradation (Kraft and Martens, 2012)
We asked if autophagic cargo receptors could interact with the Atg12~Atg5-Atg16 E3-like complex and thereby recruit it to the cargo
Employing the M-Track assay, which is based on the methylation of the human histone 3 N-terminus by the human SUV39H1 methyltransferase when the two components come into close contact (Brezovich et al, 2015; Zuzuarregui et al, 2012), we confirmed that Atg19 and the Atg12~Atg5-Atg16 complex are in close proximity in living cells (Figure 1E)
Summary
Macro-autophagy (hereafter autophagy) is a conserved pathway for the delivery of cytoplasmic material into the lysosomal system for degradation (Kraft and Martens, 2012). Upon closure of the isolation membranes, autophagosomes are formed within which the cargo is isolated from the rest of the cytoplasm. Autophagosomes subsequently fuse with the lysosomal compartment where the inner membrane and the cargo are eventually degraded. Quantification of microscopy GFP-TRAP experiments (Figure 1G) was performed using ImageJ Software. Intensity values of 30 pixels along the rim of each bead (oval selected in the GFP-channel but values measured in the mCherry-channel) were measured with Oval_Profile.java plugin, averaged and the background from a representative empty area in the image was subtracted. Beads were washed three times with IP wash buffer and resuspended in 15 mL
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