RESUMEN
J-domain proteins (JDPs) are critical components of the cellular protein quality control machinery, playing crucial roles in preventing the formation and, solubilization of cytotoxic protein aggregates. Bacteria, yeast, and plants additionally have large, multimeric heat shock protein 100 (Hsp100)-class disaggregases that resolubilize protein aggregates. JDPs interact with aggregated proteins and specify the aggregate-remodeling activities of Hsp70s and Hsp100s. However, the aggregate-remodeling properties of plant JDPs are not well understood. Here we identify eight orthologs of Sis1 (an evolutionarily conserved Class II JDP of budding yeast) in Arabidopsis thaliana with distinct aggregate-remodeling functionalities. Six of these JDPs associate with heat-induced protein aggregates in vivo and co-localize with Hsp101 at heat-induced protein aggregate centers. Consistent with a role in solubilizing cytotoxic protein aggregates, an atDjB3 mutant had defects in both solubilizing heat-induced aggregates and acquired thermotolerance as compared with wild-type seedlings. Next, we used yeast prions as protein aggregate models to show that the six JDPs have distinct aggregate-remodeling properties. Results presented in this study, as well as findings from phylogenetic analysis, demonstrate that plants harbor multiple, evolutionarily conserved JDPs with capacity to process a variety of protein aggregate conformers induced by heat and other stressors.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/metabolismo , Filogenia , Agregado de ProteínasRESUMEN
Soluble proteins that bind membranes function in numerous cellular pathways yet facile, sensitive and quantitative methods that complement and improve sensitivity of widely used liposomes-based assays remain unavailable. Here, we describe the utility of a photoactivable fluorescent lipid as a generic reporter of protein-membrane interactions. When incorporated into liposomes and exposed to ultraviolet (UV), proteins bound to liposomes become crosslinked with the fluorescent lipid and can be readily detected and quantitated by in-gel fluorescence analysis. This modification obviates the requirement for high-speed centrifugation spins common to most liposome-binding assays. We refer to this assay as Proximity-based Labeling of Membrane-Associated Proteins (PLiMAP).