The thiazide-sensitive NaCl cotransporter is targeted for chaperone-dependent endoplasmic reticulum-associated degradation.
The thiazide-sensitive NaCl cotransporter (NCC, SLC12A3) mediates salt reabsorption in the distal nephron of the kidney and is the target of thiazide diuretics, which are commonly prescribed to treat hypertension. Mutations in NCC also give rise to Gitelman syndrome, a hereditary salt-wasting disorder thought in most cases to arise from impaired NCC biogenesis through enhanced endoplasmic reticulum-associated degradation (ERAD). Because the machinery that mediates NCC quality control is completely undefined, we employed yeast as a model heterologous expression system to identify factors involved in NCC degradation. We confirmed that NCC was a bona fide ERAD substrate in yeast, as the majority of NCC polypeptide was integrated into ER membranes, and its turnover rate was sensitive to proteasome inhibition. NCC degradation was primarily dependent on the ER membrane-associated E3 ubiquitin ligase Hrd1. Whereas several ER luminal chaperones were dispensable for NCC ERAD, NCC ubiquitination and degradation required the activity of Ssa1, a cytoplasmic Hsp70 chaperone. Compatible findings were observed when NCC was expressed in mammalian kidney cells, as the cotransporter was polyubiquitinated and degraded by the proteasome, and mammalian cytoplasmic Hsp70 (Hsp72) coexpression stimulated the degradation of newly synthesized NCC. Hsp70 also preferentially associated with the ER-localized NCC glycosylated species, indicating that cytoplasmic Hsp70 plays a critical role in selecting immature forms of NCC for ERAD. Together, these results provide the first survey of components involved in the ERAD of a mammalian SLC12 cation chloride cotransporter and provide a framework for future studies on NCC ER quality control.
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
Patrick G
PG
Needham
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
Kasia
K
Mikoluk
Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
Pradeep
P
Dhakarwal
Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, Pennsylvania 15261.
Shaheen
S
Khadem
Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
Avin C
AC
Snyder
Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, Pennsylvania 15261. Electronic address: ars129@pitt.edu.
Arohan R
AR
Subramanya
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
Jeffrey L
JL
Brodsky
D000818:Animals; D001692:Biological Transport; D002412:Cations; D002460:Cell Line; D002712:Chlorides / Q000737:chemistry; D003513:Cycloheximide / Q000494:pharmacology; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D057809:HEK293 Cells; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D006801:Humans; D051379:Mice; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry; D011487:Protein Conformation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry*; D049971:Thiazides / Q000737:chemistry*; D025801:Ubiquitin / Q000737:chemistry
D002412:Cations; D002712:Chlorides; D018840:HSP70 Heat-Shock Proteins; D011500:Protein Synthesis Inhibitors; D049971:Thiazides; D025801:Ubiquitin; D012965:Sodium Chloride; D003513:Cycloheximide; D046988:Proteasome Endopeptidase Complex