Subcellular localization and detergent solubility of MVP17/rMAL, a lipid raft-associated protein in oligodendrocytes and myelin.
Detergent-insoluble, glycosphingolipid-cholesterol-enriched microdomains (lipid rafts) have been implicated in both protein trafficking and signal transduction. Previously we identified in oligodendrocytes and myelin the lipid raft-associated, integral membrane protein myelin vesicular protein of 17 kDa (MVP17)/rMAL. Here we have examined the subcellular localization and/or detergent insolubility of native and recombinant MVP17/rMAL in transfected oligodendrocytes and COS-7 cells and purified myelin. Consistent with our previous report regarding the insolubility of MVP17/rMAL in the zwitterionic detergent 3-[(3-chloramidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS), MVP17/rMAL from purified myelin and oligodendrocytes in culture was mostly insoluble upon extraction at 4 degrees C with the non-ionic detergent Triton X-100 and floated to a low density in sucrose gradient ultracentrifugation, but became detergent soluble at 37 degrees C. Data obtained by immunofluorescence microscopy of the expression of epitope-tagged MVP17/rMAL transfected into oligodendrocytes and COS-7 cells were consistent with a model in which both the N- and C-termini of this protein face the cytoplasm. Mutational analysis identified domains of MVP17/rMAL important for its subcellular localization and for its detergent solubility profile. In particular, insertional mutagenesis of loop II prevented the insertion of the mutant protein into the plasma membrane of COS-7 cells and rendered it insoluble in TX-100. Expression of full-length constructs of MVP17/rMAL in COS-7 cells resulted in an enlargement of transfected COS-7 cells, consistent with a proposed role of rMAL in vesicular trafficking.
Department of Neuroscience, University of Connecticut School of Medicine, Farmington, Connecticut 06030-3401, USA.
Taeyoon
T
Kim
Steven E
SE
Pfeiffer
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry; D018929:Cell Culture Techniques; D003902:Detergents; D015180:Electrophoresis, Gel, Two-Dimensional; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins; D021962:Membrane Microdomains / Q000737:chemistry*; D026901:Membrane Transport Proteins; D009185:Myelin Proteins; D009186:Myelin Sheath / Q000737:chemistry*; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins; D009836:Oligodendroglia / Q000737:chemistry*; D011510:Proteolipids / Q000032:analysis / Q000737:chemistry; D051381:Rats; D017472:Receptors, Interleukin-1; D012995:Solubility
D002352:Carrier Proteins; D003902:Detergents; C569028:Mal protein, rat; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009185:Myelin Proteins; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins; D011510:Proteolipids; D017472:Receptors, Interleukin-1