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

Fan Wu,Yuping Li,Fuxin Wang,Nobuo N Noda,Hong Zhang

doi:10.1074/jbc.m112.365676

Abstract

The presence of multiple homologues of the same yeast Atg protein endows an additional layer of complexity on the autophagy pathway in higher eukaryotes. The physiological function of the individual genes, however, remains largely unknown. Here we investigated the role of the two Caenorhabditis elegans homologues of the cysteine protease Atg4 in the pathway responsible for degradation of protein aggregates. Loss of atg-4.1 activity causes defective degradation of a variety of protein aggregates, whereas atg-4.2 mutants remove these substrates normally. LGG-1 precursors accumulate in atg-4.1 mutants, but not atg-4.2 mutants. LGG-1 puncta, formation of which depends on lipidation of LGG-1, are present in atg-4.1 and atg-4.2 single mutants, but are completely absent in atg-4.1; atg-4.2 double mutants. In vitro enzymatic analysis revealed that ATG-4.1 processes LGG-1 precursors about 100-fold more efficiently than ATG-4.2. Expression of a mutant form LGG-1, which mimics the processed precursor, rescues the defective autophagic degradation of protein aggregates in atg-4.1 mutants and, to a lesser extent, in atg-4.1; atg-4.2 double mutants. Our study reveals that ATG-4.1 and ATG-4.2 are functionally redundant yet display differential LGG-1 processing and deconjugating activity in the aggrephagy pathway in C. elegans.

Highlights

The function of multiple homologues of Atg4 in higher eukaryotes remains largely unknown
Overexpression of the processed form of LGG-1 rescues the defective degradation of protein aggregates in atg-4.1 mutants and, to a lesser extent, in atg-4.1; atg-4.2 double mutants
The cleaved form of LGG-1 (LGG-1-I), which lacks the final seven residues at its C terminus, was separated from the full-length LGG-1 precursor by SDS-PAGE. We found that both ATG-4.1 and ATG-4.2 were able to cleave full-length LGG-1, generating the shorter LGG1-I in the assay (Fig. 4A)

Summary

Background

The function of multiple homologues of Atg in higher eukaryotes remains largely unknown. Results: We demonstrated that the two Atg homologues in Caenorhabditis elegans have differential enzymatic activities in processing LGG-1/Atg and exhibit functional redundancy. Expression of a mutant form LGG-1, which mimics the processed precursor, rescues the defective autophagic degradation of protein aggregates in atg-4.1 mutants and, to a lesser extent, in atg-4.1; atg-4.2 double mutants. Our study reveals that ATG-4.1 and ATG-4.2 are functionally redundant yet display differential LGG-1 processing and deconjugating activity in the aggrephagy pathway in C. elegans. Mice deficient in Atg, Atg, Atg, and Atg16L show severe defects in the autophagy pathway and die during the neonatal starvation period, mice deficient in Atg4C and Atg4B do not display any obvious abnormalities and survive the early neonatal period, suggesting that there are functional divergences and redundancies between mammalian Atg homologues (18 –24). Overexpression of the processed form of LGG-1 rescues the defective degradation of protein aggregates in atg-4.1 mutants and, to a lesser extent, in atg-4.1; atg-4.2 double mutants. Our results show that atg-4.1 and atg-4.2 have differential processing activities and contribute differentially in the aggrephagy pathway in C. elegans

EXPERIMENTAL PROCEDURES

RESULTS

Findings

DISCUSSION