title: Hsp70 and Hsp90 multichaperone complexes sequentially regulate thiazide-sensitive cotransporter endoplasmic reticulum-associated degradation and biogenesis.
abstract: The thiazide-sensitive NaCl cotransporter (NCC) is the primary mediator of salt reabsorption in the distal convoluted tubule and is a key determinant of the blood pressure set point. Given its complex topology, NCC is inefficiently processed and prone to endoplasmic reticulum (ER)-associated degradation (ERAD), although the mechanisms governing this process remain obscure. Here, we identify factors that impact the ER quality control of NCC. Analyses of NCC immunoprecipitates revealed that the cotransporter formed complexes with the core chaperones Hsp90, Hsp70, and Hsp40. Disruption of Hsp90 function accelerated NCC degradation, suggesting that Hsp90 promotes NCC folding. In addition, two cochaperones, the C terminus of Hsp70-interacting protein (CHIP) and the Hsp70/Hsp90 organizer protein, were associated with NCC. Although CHIP, an E3 ubiquitin ligase, promoted NCC ubiquitination and ERAD, the Hsp70/Hsp90 organizer protein stabilized NCC turnover, indicating that these two proteins differentially remodel the core chaperone systems to favor cotransporter degradation and biogenesis, respectively. Adjusting the folding environment in mammalian cells via reduced temperature enhanced NCC biosynthetic trafficking, increased Hsp90-NCC interaction, and diminished binding to Hsp70. In contrast, cotransporters harboring disease-causing mutations that impair NCC biogenesis failed to escape ERAD as efficiently as the wild type protein when cells were incubated at a lower temperature. Instead, these mutants interacted more strongly with Hsp70, Hsp40, and CHIP, consistent with a role for the Hsp70/Hsp40 system in selecting misfolded NCC for ERAD. Collectively, these observations indicate that Hsp70 and Hsp90 comprise two functionally distinct ER quality control checkpoints that sequentially monitor NCC biogenesis.
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Bridget F
authors.initials: BF
authors.lastname: Donnelly
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Needham
authors.forename: Avin C
authors.initials: AC
authors.lastname: Snyder
authors.forename: Ankita
authors.initials: A
authors.lastname: Roy
authors.forename: Shaheen
authors.initials: S
authors.lastname: Khadem
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Brodsky
authors.forename: Arohan R
authors.initials: AR
authors.lastname: Subramanya
mesh_terms: D000818:Animals; D002460:Cell Line; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D059748:Proteolysis; D011955:Receptors, Drug / Q000235:genetics / Q000378:metabolism*; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
chemical_list: D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D046912:Multiprotein Complexes; D011955:Receptors, Drug; C578875:Slc12a3 protein, mouse; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters; C467728:STUB1 protein, human; C492951:Stub1 protein, mouse; D044767:Ubiquitin-Protein Ligases
keywords: Chloride Transport; ER-associated Degradation; Heat Shock Protein; Hsp70; Hsp90; NCC; Sodium Transport