N-terminal palmitoylation within the appropriate amino acid environment conveys on NOS2 the ability to progress along the intracellular sorting pathways.
We have analysed the mechanism by which palmitoylation permits the progression of nitric oxide synthase 2 (NOS2) along the ER-Golgi-TGN pathway. Introduction of an additional myristoylation site at the N-terminus of NOS2 resulted in a chimera that displayed an enhanced association with the particulate fraction and with the plasma membrane but did not display increased enzymatic activity. In the absence of palmitoylation, introduction of a surrogate myristoylation site resulted in a mutant NOS2 with only 25% activity compared with the wild-type enzyme. Hence, the novel surrogate myristoyl moiety not only failed to increase NOS2 activity when introduced in a wild-type sequence environment, but was also unable to rescue the inactive phenotype of the Cys3Ser mutant. Introduction of an additional palmitoylatable Cys at position 2 of the wild-type sequence resulted in a chimera that associated to a larger degree with membranes and displayed decreased activity. Our data indicate that palmitoylation of inducible NOS at position 3 exquisitely determines its transit along the secretory pathway following a route that cannot be mimicked by a surrogate myristoylation or by a palmitate at position 2. In addition, the exit of NOS2 from the TGN and the accumulation in the cellular plasma membrane per se did not correlate with increased .NO synthesis.
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain.
Inmaculada
I
Navarro-Lérida
Alberto
A
Alvarez-Barrientos
Ignacio
I
Rodríguez-Crespo
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain; D051242:Caveolin 1 / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D016207:Cytokines / Q000494:pharmacology; D019281:Dimerization; D006056:Golgi Apparatus / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D019814:Myristic Acid / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D019308:Palmitic Acid / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D000596:Amino Acids; D051242:Caveolin 1; D016207:Cytokines; D019814:Myristic Acid; D019308:Palmitic Acid; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II