title: Monitoring autophagic flux using Ref(2)P, the Drosophila p62 ortholog.
abstract: Human p62, also known as Sequestome-1 (SQSTM1), is a multifunctional scaffold protein that contains many domains, including a Phox/Bem1P (PB1) multimerization domain, an ubiquitin-associated (UBA) domain, and a light chain 3 (LC3) recognition sequence. p62 binds ubiquitinated proteins and targets them for degradation by the proteasome. In addition, p62 directly binds LC3; this may serve as a mechanism to deliver ubiquitinated proteins for degradation by autophagy. During this process, p62 itself is degraded. The inhibition of autophagy leads to the accumulation of p62, indicating that it can be used as a marker of autophagic flux. Ref(2)P (refractory to sigma P), the Drosophila ortholog of p62, is also required for the formation of ubiquitinated protein aggregates. Ref(2)P contains a putative LC3-interacting region, and genetic inhibition of autophagy in Drosophila leads to the accumulation of Ref(2)P protein levels. Thus, like p62, Ref(2)P may serve as a marker of autophagic flux. Here we provide two procedures to examine Ref(2)P protein levels in Drosophila ovaries.
authors.affiliation: The Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia V5Z 1L3, Canada Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Lindsay
authors.initials: L
authors.lastname: DeVorkin
authors.affiliation: The Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia V5Z 1L3, Canada Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Sharon M
authors.initials: SM
authors.lastname: Gorski
mesh_terms: D000818:Animals; D001343:Autophagy / Q000502:physiology*; D015820:Cadherins / Q000378:metabolism; D004268:DNA-Binding Proteins; D004330:Drosophila; D029721:Drosophila Proteins / Q000378:metabolism*; D005260:Female; D008297:Male; D009687:Nuclear Proteins / Q000378:metabolism*; D010053:Ovary / Q000166:cytology*; D013217:Starvation
chemical_list: D015820:Cadherins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D009687:Nuclear Proteins; C080596:ref(2)P protein, Drosophila; C501889:shg protein, Drosophila