Quality control for unfolded proteins at the plasma membrane.
Cellular protein homeostasis profoundly depends on the disposal of terminally damaged polypeptides. To demonstrate the operation and elucidate the molecular basis of quality control of conformationally impaired plasma membrane (PM) proteins, we constructed CD4 chimeras containing the wild type or a temperature-sensitive bacteriophage λ domain in their cytoplasmic region. Using proteomic, biochemical, and genetic approaches, we showed that thermal unfolding of the λ domain at the PM provoked the recruitment of Hsp40/Hsc70/Hsp90 chaperones and the E2-E3 complex. Mixed-chain polyubiquitination, monitored by bioluminescence resonance energy transfer and immunoblotting, is responsible for the nonnative chimera-accelerated internalization, impaired recycling, and endosomal sorting complex required for transport-dependent lysosomal degradation. A similar paradigm prevails for mutant dopamine D4.4 and vasopressin V2 receptor removal from the PM. These results outline a peripheral proteostatic mechanism in higher eukaryotes and its potential contribution to the pathogenesis of a subset of conformational diseases.
Department of Physiology, McGill University, Montréal, Quebec, Canada.
Pirjo M
PM
Apaja
Haijin
H
Xu
Gergely L
GL
Lukacs
D000818:Animals; D010582:Bacteriophage lambda / Q000737:chemistry; D015704:CD4 Antigens / Q000737:chemistry / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism*; D002522:Chlorocebus aethiops; D047369:Chromatin Immunoprecipitation; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D057146:Multivesicular Bodies / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D058767:Protein Unfolding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D054875:Ubiquitination
D015704:CD4 Antigens; D056827:Endosomal Sorting Complexes Required for Transport; D018832:Molecular Chaperones; D043562:Receptors, G-Protein-Coupled; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins