DANIOTTI JOSE LUIS
Congresos y reuniones científicas
Título:
Physico-chemical properties of lipidated sequences in membrane association and intracellular transport of peripheral proteins
Autor/es:
GUILLERMO A. GOMEZ; ALEJANDRA TRENCHI; JOSE L. DANIOTTI
Lugar:
Janelia Farm, HHMI, Virginia, USA
Reunión:
Workshop; Novel Approaches to Bio-Imaging; 2008
Institución organizadora:
Howard Hughes Medical Institute (HHMI)
Resumen:
Lipidated proteins or lipoproteins participate in a plethora of cellular events, such as signal transduction, vesicular transport and cytosqueleton dynamics. At difference of transmembrane proteins, many lipoproteins associate to membranes due to the hydrophobic characteristics and number of lipid anchors, and physicochemical properties of amino acid around these modifications. However the mechanisms of subcellular distribution, inclusion in specific vesicular intermediates and lateral segregation (sorting events), remains unknown. In our lab, we are studying such events utilizing GFP fused to different sequences, which are target of different lipid modifications. GAP-43, H-Ras and K-Ras, where employed as models of lipoproteins, by virtue of the different characteristics of lipid modifications and amino-acid sequence around these. The C-terminal domain of H-Ras is farnesylated and doubly acylated, the C-terminal domain of K-Ras is farnesylated but not acylated and the N-terminal domain of GAP-43 is dually acylated. Aditionally, there is a polybasic domain around farnesylated cysteine in K-Ras (q=+6) and acylated cysteines in GAP-43 (q=+3). These proteins are located mainly at the plasma membrane and recycling endosomes, when expressed in CHO-K1 cells. Our studies point the endosomal localization of these proteins, and demonstrate that it is due at least by two different mechanisms which is dependent of the lipidated sequence, as we determined by characterization of membrane binding properties, hidrophobicity characteristics and dynamics of recycling endosome localization. Acylated proteins reach the recycling endosome compartment trough vesicular intermediates, meanwhile, K-Ras targets to these endosomal membranes by a desorption-reabsortion mechanism, driven by a electrostatic gradient between plasma membrane and endomembranes. We also demonstrate that inner leaflet surface membrane potential due to poliphosphoinositides at the plasma membrane, is the major contribution to the subcellular distribution of K-Ras. Our results suggest that physicochemical properties of lipid modifications and sequences around it, regulate, the functions of these proteins in two ways: the chemical composition of membranes which these are bounded, and the dynamics of membrane association, which are relevant for proper protein-protein or protein-lipid interactions at the membrane interphase.
