Mutational analysis identifies a short atypical membrane attachment sequence (KYWFYR) within caveolin-1.
Caveolae are vesicular invaginations of the plasma membrane. Their formation is strictly dependent on the expression of the caveolin coat proteins. During transit to the plasma membrane, approximately 15 monomers of caveolin-1 assemble into a multivalent homo-oligomer. Caveolae are most likely generated through the subsequent interaction of these caveolin homo-oligomers with one another, with sphingolipids, and with cholesterol. Membrane association of caveolin-1 is critical to this process and is facilitated by an atypical N-terminal membrane attachment domain (residues 82-101), termed N-MAD. To better understand the membrane attachment function of N-MAD, we performed a detailed mutational analysis of the 20 amino acid N-MAD peptide sequence fused to the C-terminus of the soluble reporter green fluorescent protein (GFP). Removal of the distal six residues (KYWFYR) within N-MAD prevents membrane attachment in cells as assessed by hypotonic lysis, detergent solubility, carbonate extraction, and fluorescence microscopy. These six residues (KYWFYR) are sufficient to confer membrane attachment to GFP, an otherwise soluble protein. Both the central aromatic and flanking basic residues in this sequence are required for membrane attachment, as the sequence YWFY does not confer membrane affinity to GFP. Although the KYWFYR sequence within N-MAD facilitates membrane association, we show that the entire N-MAD sequence is required for targeting to lipid rafts/caveolae.
Woodman
Scott E
SE
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Schlegel
Amnon
A
Cohen
Alex W
AW
Lisanti
Michael P
MP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D051242:Caveolin 1; D022461:Caveolins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D016296:Mutagenesis
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins