Browsing by MeSH term "Golgi Apparatus"
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Publication Distance-dependent cellular palmitoylation of de-novo-designed sequences and their translocation to plasma membrane subdomains.(The Company of Biologists, 2002-08-01) Navarro-Lerida, Inmaculada; Alvarez-Barrientos, Alberto; Gavilanes, Francisco; Rodriguez-Crespo, Ignacio; Comunidad de Madrid (España); Spanish DGIUsing 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.Publication N-terminal palmitoylation within the appropriate amino acid environment conveys on NOS2 the ability to progress along the intracellular sorting pathways.(The Company of Biologists, 2006-04-15) Navarro-Lerida, Inmaculada; Alvarez-Barrientos, Alberto; Rodriguez-Crespo, IgnacioWe 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.Publication The Calcineurin Variant CnAβ1 Controls Mouse Embryonic Stem Cell Differentiation by Directing mTORC2 Membrane Localization and Activation(Elsevier, 2016-11-17) Gomez-Salinero, Jesus M.; Lopez-Olaneta, Marina; Ortiz-Sanchez, Paula; Larrasa-Alonso, Javier; Gatto, Alberto; Felkin, Leanne E; Barton, Paul J R; Navarro-Lerida, Inmaculada; del Pozo, Miguel Angel; García-Pavía, Pablo; Sundararaman, Balaji; Giovinazzo, Giovanna; Yeo, Gene W; Lara-Pezzi, Enrique; Unión Europea. Comisión Europea; Ministerio de Ciencia e Innovación (España); Comunidad de Madrid (España); Ministerio de Economía y Competitividad (España); Fundación ProCNICEmbryonic stem cells (ESC) have the potential to generate all the cell lineages that form the body. However, the molecular mechanisms underlying ESC differentiation and especially the role of alternative splicing in this process remain poorly understood. Here, we show that the alternative splicing regulator MBNL1 promotes generation of the atypical calcineurin Aβ variant CnAβ1 in mouse ESCs (mESC). CnAβ1 has a unique C-terminal domain that drives its localization mainly to the Golgi apparatus by interacting with Cog8. CnAβ1 regulates the intracellular localization and activation of the mTORC2 complex. CnAβ1 knockdown results in delocalization of mTORC2 from the membrane to the cytoplasm, inactivation of the AKT/GSK3β/β-catenin signaling pathway, and defective mesoderm specification. In summary, here we unveil the structural basis for the mechanism of action of CnAβ1 and its role in the differentiation of mESCs to the mesodermal lineage.