The role of an intracellular cysteine stretch in the sorting of the type II Na/phosphate cotransporter.
The type II Na/phosphate cotransporters (NaPi-II) are critical for the control of plasma phosphate levels in vertebrates. NaPi-IIb mediates phosphate uptake from the small intestine followed by glomerular filtration and selective reabsorption from the renal proximal tubule by NaPi-IIa and NaPi-IIc. A C-terminal stretch of cysteine residues represents the hallmark of the NaPi-IIb isoforms. This motif is well conserved among NaPi-IIb type transporters but not found in other membrane proteins. To investigate the role of this motif we analyzed NaPi-II constructs in transiently and stably transfected MDCK cells. This cell line targets the NaPi-IIb isoforms from flounder and mouse to the apical membrane whereas the mouse IIa isoform shows no plasma membrane preference. Different parts of mouse NaPi-IIa and NaPi-IIb C-termini were fused to GFP-tagged flounder NaPi-II. The constructs showed strong staining of the plasma membrane with NaPi-IIb related constructs sorted predominantly apically, the IIa constructs localized apically and basolaterally with slight intracellular retention. When the cysteine stretch was inserted into the NaPi-IIa C-terminus, the construct was retained in a cytoplasmic compartment. 2-bromopalmitate, a specific palmitoylation inhibitor, released the transporter to apical and basolateral membranes. The drug also leads to a redistribution of the NaPi-IIb construct to both plasma membrane compartments. Immunoprecipitation of tagged NaPi-II constructs from [(3)H]-palmitate labeled MDCK cells indicated that the cysteine stretch is palmitoylated. Our results suggest that the modified cysteine motif prevents the constructs from basolateral sorting. Additional sorting determinants located downstream of the cysteine stretch may release the cargo to the apical compartment.
McHaffie
Gavin S
GS
Epithelial Research Group, Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
Graham
Chris
C
Kohl
Beate
B
Strunck-Warnecke
Ursula
U
Werner
Andreas
A
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism*; D004285:Dogs; D005432:Flounder; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D050610:Sodium-Phosphate Cotransporter Proteins, Type II / Q000737:chemistry* / Q000378:metabolism*; D013045:Species Specificity; D013329:Structure-Activity Relationship
D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D003545:Cysteine