A Single Protease, Apg4B, Is Specific for the Autophagy-related Ubiquitin-like Proteins GATE-16, MAP1-LC3, GABARAP, and Apg8L

Abstract

Apg8 is a ubiquitin-like protein involved in autophagy in yeast. Apg8 is covalently but transiently attached to membrane lipids through the actions of activating, conjugating, and processing/deconjugating enzymes. The mammalian Apg8 homologues GATE-16, GARARAP, and MAP1-LC3 have been implicated in intra-Golgi transport, receptor sorting, and autophagy, respectively. All are served by a single set of activating and conjugating enzymes. Here we identify a novel mammalian Apg8 homologue, which we name Apg8L, and describe the synthesis of electrophilic probes based on the GATE-16, GARARAP, MAP1-LC3, and Apg8L proteins. These probes not only form specific adducts in crude cell lysates, but also allow identification of the cellular proteases specific for the C termini of these Apg8 homologues. We find a single protease, Apg4B/autophagin-1, capable of acting on GATE-16, GABARAP, MAP1-LC3, and Apg8L. The Apg4B/autophagin-1 protease thus serves as a processing/deconjugating enzyme for these four highly divergent mammalian Apg8 homologues.

Differential Function of the Two Atg4 Homologues in the Aggrephagy Pathway in Caenorhabditis elegans

“…The multiple homologues might act redundantly, function in a tissue-specific fashion, or be expressed at different developmental stages; alternatively they may have evolved to accomplish other functions distinct from autophagy. The substrate preferences of four mammalian Atg4s toward different Atg8s have been characterized in vitro (11,13,14,17). HsAtg4B cleaves all Atg8s (13)(14)(15).…”

“…The substrate preferences of four mammalian Atg4s toward different Atg8s have been characterized in vitro (11,13,14,17). HsAtg4B cleaves all Atg8s (13)(14)(15). HsAtg4A efficiently cleaves GATE-16, but not other Atg8s, while HsAtg4C and HsAtg4D both have minimal activity (11,17).…”

Vesicular trafficking and autophagosome formation

“…10 There are four members of the Atg4 family, Atg4a-d, also called the autophagins1-4, 11 and among these Atg4b seems to have the broadest specificity. [12][13][14] The Atg4b-dependent cleavage of LC3 requires the catalytic residue, Cys74, and mutation of this residue has been shown to inhibit the cleavage of LC3-II and GABARAP-II in vitro and in vivo in cell culture model systems. Overexpression of Atg4b was also found to inhibit the lipidation of LC3, 14 and the formation of GFP-LC3-positive autophagosomes.…”

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism

“…2 LC3 is cotranslationally processed by ATG4B, a cysteine protease, to generate LC3B-I. 3 ATG4B action exposes a glycine at the carboxyl terminus of LC3B-I, which is activated by ATG7 and ATG3 and transferred to phosphatidylethanolamine to produce LC3B-II. 4 LC3B-II lies on both surfaces of autophagosome (external and internal) where it serves to elongate the membrane and recruit the cargo.…”