Mammalian red cell membrane Rh polypeptides are selectively palmitoylated subunits of a macromolecular complex.

S Hartel-Schenk,P Agre

doi:10.1016/s0021-9258(18)42803-7

S Hartel-Schenk, P Agre

Open Access

https://doi.org/10.1016/s0021-9258(18)42803-7

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Abstract

Incubation of [3H]palmitic acid, ATP, and CoA with inside-out membrane vesicles prepared from human or other mammalian red cells resulted in nearly exclusive 3H-palmitoylation of the Mr = 32,000 Rh polypeptides. [3H]Palmitic, [3H]myristic, and [3H]oleic acids were comparably esterified onto Rh polypeptides in inside-out membrane vesicles in the presence of ATP and CoA, although [3H]palmitic acid was preferentially incorporated by intact human red cells. Experiments using sulfhydryl reagents or tryptic digestions suggested that multiple sulfhydryl groups on the Rh polypeptides located near the cytoplasmic leaflet of the lipid bilayer were 3H-palmitoylated; the exofacial sulfhydryl group essential for Rh antigenic reactivity was not 3H-palmitoylated. Transfer of fatty acid from [14C]palmitoyl-CoA to sites on the Rh polypeptides occurred even after previous incubation of inside-out membrane vesicles at 95 degrees C or after solubilization of inside-out membrane vesicles in Triton X-100. Hydrodynamic analyses of Triton X-100-solubilized membranes surprisingly demonstrated that 3H-palmitoylated Rh polypeptides behaved as a protein of apparent Mr = 170,000. These in vitro studies suggest that palmitoylation of Rh polypeptides occurs within a macromolecular complex by a highly selective but possibly nonenzymatic mechanism.

Highlights

Incubation of [‘Hlpalmitic acid, ATP, andCoA with Rh polypeptides are major palmitoylated components of inside-outmembranevesicles prepared from humaonr human red cell membranes, and other mammalian redcells resulted in nearleyxclusive palmitoylation of cellular proteins is recognized to be a spe
Experiments using sulfhydrylreagents or trypticdigestionssuggested that multiple sulfhydryl groupons the Rh polypeptides located near the cytoplasmliecaflet of the lipid bilayer evolutionary, and functional significance of fatty acylation remain unknown
It is likely that thisvalue reflects imprecisions inherent in determining the physical size of a hydrophobic membrane protein;each of the analyses suggest that the Rhpolypeptides exist in the membrane as a macromolecular complex

Summary

Total Phospholipid Total Phospholipid

Human red cells (RBC) were incubated with ['Hlpalmitic acid, ["Hlmyristic acid, or [3H]oleicacid (10 pCi/nmol). Following 15-h incubations at 37 "C, the red cell membranes and vesicles were analyzed by SDS-PAGE fluorography (see "Experimental Procedures"). Intact human red cells were incubated 15 h at 37 "C with [3H] fatty acids (10 pCi/nmol). The red cells were washed, and cell membrane retained radioactivity was measured (see "Experimental Procedures"). Human red cells were incubated with [3H]fatty acids as described above and divided into aliquots. Inside-out membrane vesicles were prepared from the second aliquot of red cells, and these vesicles were back-extracted as described above but without 0.15 M NaCl. Control experiments showed that 4 0 % of radioactivity was back-extracted when albumin was omitted. Lipids were extracted from membranes in chloroform/methanol and phospholipids were isolated by thin layer chromatography (Staufenbiel, 1988) using the buffer of Schmidt et al (1979)

Palmitoylated Rh Polypeptide Complex

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