The role of proline in the membrane re-entrant helix of caveolin-1.
Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103-122. We have performed an in silico analysis of the conformational preference of this segment of caveolin-1 using PepLook. We find that there is one main group of stable conformations corresponding to a hydrophobic U bent model that would not traverse the membrane. Furthermore, the calculations predict that substituting the Pro(110) residue with an Ala will change the conformation to a straight hydrophobic helix that would traverse the membrane. We have expressed the P110A mutant of caveolin-1, with a FLAG tag at the N terminus, in HEK 293 cells. We evaluate the topology of the proteins with confocal immunofluorescence microscopy in these cells. We find that FLAG tag at the N terminus of the wild type caveolin-1 is not reactive with antibodies unless the cell membrane is permeabilized with detergent. This indicates that in these cells, the hydrophobic segment of this protein is not transmembrane but takes up a bent conformation, making the protein monotopic. In contrast, the FLAG tag at the N terminus of the P110A mutant is equally exposed to antibodies, before and after membrane permeabilization. We also find that the P110A mutation causes a large reduction of endocytosis of caveolae, cellular lipid accumulation, and lipid droplet formulation. In addition, we find that this mutation markedly reduces the ability of caveolin-1 to form structures with the characteristic morphology of caveolae or to partition into the detergent-resistant membranes of these cells. Thus, the single Pro residue in the membrane-inserting segment of caveolin-1 plays an important role in both the membrane topology and localization of the protein as well as its functions.
Aoki
Satoko
S
From the Department of Biochemistry and Biomedical Sciences, McMaster University Health Science Center, Hamilton, Ontario L8N 3Z5, Canada.
Thomas
Annick
A
Centre de Biophysique Moléculaire Numérique, ULg Gembloux Agro-BioTech, Passage des Déportés 2, 5030 Gembloux, Belgium.
Decaffmeyer
Marc
M
Centre de Biophysique Moléculaire Numérique, ULg Gembloux Agro-BioTech, Passage des Déportés 2, 5030 Gembloux, Belgium.
Brasseur
Robert
R
Centre de Biophysique Moléculaire Numérique, ULg Gembloux Agro-BioTech, Passage des Déportés 2, 5030 Gembloux, Belgium.
Epand
Richard M
RM
From the Department of Biochemistry and Biomedical Sciences, McMaster University Health Science Center, Hamilton, Ontario L8N 3Z5, Canada. Electronic address: epand@mcmaster.ca.
D019943:Amino Acid Substitution; D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D051379:Mice; D020125:Mutation, Missense; D011392:Proline / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary
D051242:Caveolin 1; D011392:Proline