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Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells
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Golgi Alpha1,2-Mannosidase IA Promotes Efficient Endoplasmic Reticulum-Associated Degradation of NKCC2

Sylvie Demaretz1,2, Elie Seaayfan1,2, Dalal Bakhos-Douaihy1,2

  • 1Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, F-75006 Paris, France.

Cells
|January 11, 2022
PubMed

Insights

Golgi alpha1, 2-mannosidase IA (ManIA) targets misfolded kidney Na-K-2Cl cotransporter (NKCC2) for degradation. This ManIA-mediated pathway enhances ER-associated degradation (ERAD) of NKCC2 mutants, crucial for type I Bartter syndrome.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Renal Physiology

Background:

  • Mutations in NKCC2 cause type I Bartter syndrome, a severe kidney disorder.
  • ER transport is a rate-limiting step for NKCC2 maturation.
  • ER quality control mechanisms for NKCC2 and its mutants are poorly understood.

Purpose of the Study:

  • Identify novel ER quality control components interacting with NKCC2.
  • Investigate the role of Golgi alpha1, 2-mannosidase IA (ManIA) in NKCC2 maturation and degradation.
  • Elucidate the mechanism of ManIA-mediated ER-associated degradation (ERAD) of NKCC2.

Main Methods:

  • Co-immunoprecipitation to identify binding partners.
  • Western blotting to assess protein abundance.
  • Cycloheximide chase assays to study protein stability.
  • Mutagenesis to investigate functional domains of ManIA.
  • Treatment with proteasome inhibitors and mannosidase inhibitors.

Main Results:

  • ManIA binds to immature NKCC2 at the cis-Golgi network.
  • ManIA coexpression reduces total NKCC2 levels, particularly folding mutants.
  • ManIA accelerates NKCC2 degradation via the proteasome pathway.
  • ManIA's catalytic activity and cytoplasmic tail are essential for its effect on NKCC2 maturation.

Conclusions:

  • ManIA is a novel binding partner of NKCC2.
  • ManIA mediates an ERAD pathway that promotes the degradation of misfolded NKCC2.
  • This pathway involves mannose trimming and proteasomal degradation.
  • ManIA plays a critical role in renal cell quality control of NKCC2, impacting Bartter syndrome pathogenesis.
Keywords:
Bartter syndromeERADGolgiNKCC2alpha1,2-mannosidase IAhypertensionkidneymembraneprotein quality controltrafficking

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