A conserved hydrophobic tetrad near the C terminus of the secretory Na+-K+-2Cl- cotransporter (NKCC1) is required for its correct intracellular processing.
Little is known about the intracellular folding and trafficking of integral membrane proteins. Here we identify a hydrophobic amino acid tetrad (ILLV) close to the C terminus of the secretory Na+-K+-2Cl- cotransporter (NKCC1) that is important for the proper intracellular processing of this protein. This tetrad appears in a C-terminal sequence pattern that is conserved across species in a number of members of the NKCC1 gene family (slc12) of electroneutral salt transporters. We studied the effects of various mutations of these amino acids on NKCC1 transiently transfected into HEK-293 cells. Our results show that mutation of two of these residues to alanine leads to a >50% reduction in expression and complex glycosylation levels and that multiple mutations to alanine have cumulative effects. By contrast, scrambling of these amino acids, or mutation of other nearby conserved C-terminal residues, has little effect on these parameters. Mutation of ILLV to AAAA reduces complex glycosylation of NKCC1 by approximately 90% and results in a protein that does not form stable dimers and is retained in the endoplasmic reticulum in a highly aggregated state. Our results are consistent with the hypothesis that mutation of the hydrophobic tetrad ILLV to AAAA leads to the ab initio misfolding and concomitant aggregation of this NKCC1 mutant, resulting in its retention in the endoplasmic reticulum.
Nezu
Akihiro
A
Membrane Biology Section, Molecular Physiology and Therapeutics Branch, NIDCR, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892-1190, USA.
Parvin
Most Nahid
MN
Turner
R James
RJ
D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D006031:Glycosylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D020125:Mutation, Missense; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2
C578877:SLC12A2 protein, human; C578879:Slc12a2 protein, rat; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2