Isolation and characterization of novel plekhm1 and def8 mutant alleles in Drosophila

Abstract

Lysosomal degradation of cytoplasmic components by autophagy ensures the continuous turnover of proteins and organelles and aids cellular survival during nutrient deprivation and other stress conditions. Lysosomal targeting of cytoplasmic proteins and organelles requires the concerted action of several proteins and multisubunit complexes. The core components of this machinery are conserved from yeast to humans and many of them are well-characterized; however, novel molecular players have been recently discovered and are waiting for detailed analysis. The osteopetrosis-linked PLEKHM1 protein is a lysosomal adaptor involved in autophagosome and endosome to lysosome fusion events and its role in lysosomal positioning in osteoclasts was reported together with its proposed binding partner, the relatively uncharacterized DEF8 protein. Here, we report the generation and subsequent analysis of novel mutant alleles of Drosophila plekhm1 and def8. Interestingly, the CRISPR-generated null mutations of these genes do not have any obvious effects on autophagy in Drosophila tissues, even though RNAi knockdown of these genes seems to perturb autophagy. Although these results are quite surprising and raise the possibility of compensatory changes in the case of null mutants, the new alleles will be valuable tools in future studies to understand the cellular functions of Drosophila Plekhm1 and Def8 proteins.

The autophagy protein Def8 is altered in Alzheimer's disease and Aβ42-expressing Drosophila brains

“…Human and Drosophila proteins highlighted domain and sequence conservation, with Def8 positive immunoreactivity in flies, which allowed us to establish a structural homology in both species. Previous reports confirmed a strong link between Def8 and other autophagy components, Rab7a and Plekhm1 21 , 44 . Our network analyses also confirmed those observations in Drosophila .…”

Molecular Mechanisms of Craniofacial and Dental Abnormalities in Osteopetrosis

“…Most research supports the idea that mammalian PLEKHM1 binds to Rab7, the HOPS complex, and GABARAP (gamma-aminobutyric acid receptor-associated) family proteins, which promotes the fusion of lysosomes with late endosomes and autophagosomes [ 1 ]. Surprisingly, Maruzs et al [ 82 ] reported that the novel mutant alleles of Drosophila, plekhm1 and def8 , do not have obvious influences on autophagy in Drosophila.…”