Functional characterization of a vacuolar invertase from Solanum lycopersicum: Post-translational regulation by N-glycosylation and a proteinaceous inhibitor

Alexandra Tauzin (1) , Gerlind Sulzenbacher (2) , Mickael Lafond (3) , Véronique Desseaux (4) , Ida Barbara Reca (5) , Josette Perrier (3) , Daniela Bellincampi (5) , Patrick Fourquet (6) , Christian Levêque (7) , Thierry Giardina (3)

1 TWB - Toulouse White Biotechnology
2 AFMB - Architecture et fonction des macromolécules biologiques
3 ISM2 - Institut des Sciences Moléculaires de Marseille
4 PNP - Physiologie neurovégétative - PNV
5 Dipartimento di Biologia e Biotecnologie ''Charles Darwin''
6 CIML - Centre d'Immunologie de Marseille - Luminy
7 ORSTOM

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 muta-genesis. 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 thermosta-bility. 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 het-erologously 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.

Mots clés: Vacuolar invertase; Proteinaceous inhibitor; N-Glycosylation; Surface plasmon resonance; Solanum lycopersicum; Proteineprotein interactions

Domaines: Biochimie [q-bio.BM]; Botanique

Alexandra Tauzin, Gerlind Sulzenbacher, Mickael Lafond, Véronique Desseaux, Ida Barbara Reca, et al.. Functional characterization of a vacuolar invertase from Solanum lycopersicum: Post-translational regulation by N-glycosylation and a proteinaceous inhibitor. Biochimie, 2014, 101, pp.39-49. ⟨10.1016/j.biochi.2013.12.013⟩. ⟨hal-01793351⟩