title: Functional characterization of a vacuolar invertase from Solanum lycopersicum: post-translational regulation by N-glycosylation and a proteinaceous inhibitor.
abstract: Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyse sucrose into glucose and fructose. The cDNA encoding a vacuolar invertase from Solanum lycopersicum (TIV-1) was cloned and heterologously expressed in Pichia pastoris. The functional role of four N-glycosylation sites in TIV-1 has been investigated by site-directed mutagenesis. Single mutations to Asp of residues Asn52, Asn119 and Asn184, as well as the triple mutant (Asn52, Asn119 and Asn184), lead to enzymes with reduced specific invertase activity and thermostability. Expression of the N516D mutant, as well as of the quadruple mutant (N52D, N119D, N184D and N516D) could not be detected, indicating that these mutations dramatically affected the folding of the protein. Our data indicate that N-glycosylation is important for TIV-1 activity and that glycosylation of N516 is crucial for recombinant enzyme stability. Using a functional genomics approach a new vacuolar invertase inhibitor of S. lycopersicum (SolyVIF) has been identified. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli. Specific interactions between SolyVIF and TIV-1 were investigated by an enzymatic approach and surface plasmon resonance (SPR). Finally, qRT-PCR analysis of TIV-1 and SolyVIF transcript levels showed a specific tissue and developmental expression. TIV-1 was mainly expressed in flowers and both genes were expressed in senescent leaves.
authors.affiliation: Aix Marseille Université, Centrale Marseille, CNRS, iSm2 UMR 7313, 13397 Marseille, France.
authors.forename: Alexandra S
authors.initials: AS
authors.lastname: Tauzin
authors.affiliation: Aix Marseille Université, CNRS, AFMB UMR 7257, 13288 Marseille, France.
authors.forename: Gerlind
authors.initials: G
authors.lastname: Sulzenbacher
authors.affiliation: Aix Marseille Université, Centrale Marseille, CNRS, iSm2 UMR 7313, 13397 Marseille, France.
authors.forename: Mickael
authors.initials: M
authors.lastname: Lafond
authors.affiliation: Aix Marseille Université, Centrale Marseille, CNRS, iSm2 UMR 7313, 13397 Marseille, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Desseaux
authors.affiliation: Dipartimento di Biologia e Biotecnologie "Charles Darwin", "Sapienza" Università di Roma, Roma, Italy.
authors.forename: Ida Barbara
authors.initials: IB
authors.lastname: Reca
authors.affiliation: Aix Marseille Université, Centrale Marseille, CNRS, iSm2 UMR 7313, 13397 Marseille, France.
authors.forename: Josette
authors.initials: J
authors.lastname: Perrier
authors.affiliation: Dipartimento di Biologia e Biotecnologie "Charles Darwin", "Sapienza" Università di Roma, Roma, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Bellincampi
authors.affiliation: Centre d'Immunologie de Marseille Luminy, Service Protéomique, 13288 Marseille, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Fourquet
authors.affiliation: Aix Marseille Université, CNRS, Plateformes de Recherche en Neurosciences/CAPM, UMR 7286, 13344 Marseille, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Lévêque
authors.affiliation: Aix Marseille Université, Centrale Marseille, CNRS, iSm2 UMR 7313, 13397 Marseille, France. Electronic address: thierry.giardina@univ-amu.fr.
authors.forename: Thierry
authors.initials: T
authors.lastname: Giardina
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D004795:Enzyme Stability; D018506:Gene Expression Regulation, Plant; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D014617:Vacuoles / Q000201:enzymology*; D043324:beta-Fructofuranosidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D043324:beta-Fructofuranosidase
keywords: N-Glycosylation; Proteinaceous inhibitor; Protein–protein interactions; Solanum lycopersicum; Surface plasmon resonance; Vacuolar invertase