Distance-dependent cellular palmitoylation of de-novo-designed sequences and their translocation to plasma membrane subdomains.
Using recursive PCR, we created an artificial protein sequence that consists of a consensus myristoylation motif (MGCTLS) followed by the triplet AGS repeated nine times and fused to the GFP reporter. This linker-GFP sequence was utilized as a base to produce multiple mutants that were used to transfect COS-7 cells. Constructs where a 'palmitoylable' cysteine residue was progressively moved apart from the myristoylation site to positions 3, 9, 15 and 21 of the protein sequence were made, and these mutants were used to investigate the effect of protein myristoylation on subsequent palmitoylation, subcellular localization, membrane association and caveolin-1 colocalization. In all cases, dual acylation of the GFP chimeras correlated with translocation to Triton X-100-insoluble cholesterol/sphingomyelin-enriched subdomains. Whereas a strong Golgi labeling was observed in all the myristoylated chimeras, association with the plasma membrane was only observed in the dually acylated constructs. Taking into account the conflicting data regarding the existence and specificity of cellular palmitoyl-transferases, our results provide evidence that de-novo-designed sequences can be efficiently S-acylated with palmitic acid in vivo, strongly supporting the hypothesis that non-enzymatic protein palmitoylation can occur within mammalian cells. Additionally, this palmitoylation results in the translocation of the recombinant construct to low-fluidity domains in a myristate-palmitate distance-dependent manner.
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, 28040 Madrid, Spain.
Inmaculada
I
Navarro-Lérida
Alberto
A
Alvarez-Barrientos
Francisco
F
Gavilanes
Ignacio
I
Rodriguez-Crespo
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D019556:COS Cells; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002451:Cell Compartmentation / Q000235:genetics*; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000172:deficiency; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D021962:Membrane Microdomains / Q000378:metabolism; D018613:Microscopy, Confocal; D019814:Myristic Acid / Q000378:metabolism*; D017830:Octoxynol; D010168:Palmitates / Q000494:pharmacology; D019308:Palmitic Acid / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics*; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics; D012995:Solubility / Q000187:drug effects; D013109:Sphingomyelins / Q000378:metabolism
D051242:Caveolin 1; D022461:Caveolins; D008164:Luminescent Proteins; D010168:Palmitates; D011506:Proteins; D011993:Recombinant Fusion Proteins; D013109:Sphingomyelins; D019814:Myristic Acid; D049452:Green Fluorescent Proteins; C022776:2-bromopalmitate; D019308:Palmitic Acid; D017830:Octoxynol; D002784:Cholesterol