Study of the structure and the function of the UDP-GlcNAc transporter, SLC35A3
The nucleotide-sugar transporters (NSTs) are integral membrane proteins of the Golgi apparatus \nand the Endoplasmic Reticulum (ER), that controls the flux of nucleotide-sugars from the cytosol to \nthe lumen, where they serve as substrate for specific glycosyltransferases during the synthesis of \n...
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| Formato: | Tesis de maestría acceptedVersion |
| Lenguaje: | Inglés |
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Facultad de Farmacia y Bioquímica
2016
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_2811 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_2811.dir/2811.PDF |
| Aporte de: |
| Sumario: | The nucleotide-sugar transporters (NSTs) are integral membrane proteins of the Golgi apparatus \nand the Endoplasmic Reticulum (ER), that controls the flux of nucleotide-sugars from the cytosol to \nthe lumen, where they serve as substrate for specific glycosyltransferases during the synthesis of \ndiverse glycoconjugates (glycolipids, glycoproteins and proteoglycans). The NSTs are members of \nSLC35 family, a group of highly conserved hydrophobic proteins with multiple transmembrane \ndomains with a size of 30-40kDa. Mutations in these proteins, cause development anomalies in \neukaryotic organisms, from protozoa to mammals, indicating the physiological relevance of these \ntransporters. \nFor the nucleotide-sugar transport process an antiporter mechanism, nucleotide sugar/ nucleoside \nmonophosphate, have been proposed. Nevertheless the molecular basis of ligand recognition and \ntransport is unknown. Like other membrane transport proteins the progress in structure-function \nstudies are limited due to the complexity in the transport activity characterization assays and the \ndifficulty to obtain large amounts of purified protein. In this context the general aim of this work \nwas to determine the transport mechanism of NST at a molecular level, using the murine SLC35A3 \ntransporter, specific for UDP-GlcNAc as a model. Based on bioinformatics tools, GFP-Fusion \ntechnology, a complementation activity assay and cysteine scanning mutagenesis we developed an \napproach to identify and characterize residues critical for the UDP-GlcNAc transport activity by \nSLC35A3. We functionally characterized the MmSLC35A3 transporter as a UDP-GlcNAc \ntransporter based on bioinformatic tools, phenotypic correction and Cysteine Scanning Mutagenesis \nassays. Two critical residues for the MmSLC35A3 functioning, Glu47 and Lys50, localized in the \nsecond transmembrane domain were identified. On the other hand, based on the approach \ndeveloped by Newstead et al (2007), we were able to optimize the MmSLC35A3 expression and \npurification, obtaining a yield of 3.9 mg of protein per L of culture, estimating that this approach \nwill leads to a function-structure studies on MmSLC35A3 pure. This last approach will be based on the labeling of the cysteine residues on the cysteine unique mutants with fluorescents probes. The \nobjective of this label is to know which residues are exposed to the cytosolic or the luminal space |
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