N-terminal palmitoylation within the appropriate amino acid environment conveys on NOS2 the ability to progress along the intracellular sorting pathways

• Published in: Journal of cell science Vol. 119; no. Pt 8; pp. 1558 - 1569
• Main Authors: Navarro-Lérida, Inmaculada, Alvarez-Barrientos, Alberto, Rodríguez-Crespo, Ignacio
• Format: Journal Article
• Language: English
• Published: England 15.04.2006
• Subjects: Amino Acid Sequence; Amino Acids - chemistry; Animals; Catalytic Domain; Caveolin 1 - metabolism; Cell Membrane - metabolism; Cercopithecus aethiops; COS Cells; Cytokines - pharmacology; Dimerization; Golgi Apparatus - metabolism; Molecular Sequence Data; Mutation; Myristic Acid - metabolism; Nitric Oxide - pharmacology; Nitric Oxide Synthase Type II - chemistry; Nitric Oxide Synthase Type II - genetics; Nitric Oxide Synthase Type II - metabolism; Nitric Oxide Synthase Type II - physiology; Palmitic Acid - metabolism; Protein Processing, Post-Translational; Protein Transport; Sequence Homology, Amino Acid; Signal Transduction
• ISSN: 0021-9533; 1477-9137
• DOI: 10.1242/jcs.02878

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.