Site-Specific Mutagenesis by Using an Accurate Recombinant Polymerase Chain Reaction Method

Ansaldi, Mireille; Lepelletier, Michèle; Méjean, Vincent

doi:10.1006/abio.1996.0060

Cited by 87 publications

(59 citation statements)

References 2 publications

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“…The pSP2 plasmid expresses a C-terminal Jen1-GFP fusion protein under the control of the native JEN1 promoter. Mutations that substitute JEN1 lysine codons for arginine were introduced in the plasmid pSP2, using site-directed mutagenesis as described (32). The resulting plasmids pSP3 to -5 express the resulting mutant alleles of JEN1 ( Table 1).…”

Section: Methodsmentioning

confidence: 99%

Glucose-induced Ubiquitylation and Endocytosis of the Yeast Jen1 Transporter

Paiva

Vieira

Nondier

et al. 2009

Journal of Biological Chemistry

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Protein ubiquitylation is essential for many events linked to intracellular protein trafficking. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitylation remain largely unknown. Plasma membrane transporters are subjected to tightly regulated endocytosis, and ubiquitylation is a key signal at several stages of the endocytic pathway. The yeast monocarboxylate transporter Jen1 displays glucose-regulated endocytosis. We show here that casein kinase 1-dependent phosphorylation and HECT-ubiquitin ligase Rsp5-dependent ubiquitylation are required for Jen1 endocytosis. Ubiquitylation and endocytosis of Jen1 are induced within minutes in response to glucose addition. Jen1 is modified at the cell surface by oligo-ubiquitylation with ubiquitin-Lys 63 linked chain(s), and Jen1-Lys 338 is one of the target residues. UbiquitinLys 63 -linked chain(s) are also required directly or indirectly to sort Jen1 into multivesicular bodies. Jen1 is one of the few examples for which ubiquitin-Lys 63 -linked chain(s) was shown to be required for correct trafficking at two stages of endocytosis: endocytic internalization and sorting at multivesicular bodies.Ubiquitylation is one of the most prevalent protein posttranslational modifications in eukaryotes. In addition to its role in promoting proteasomal degradation of target proteins, ubiquitylation has been shown to regulate multiple processes, including DNA repair, signaling, and intracellular trafficking. Ubiquitylation serves as a key signal mediating the internalization of plasma membrane receptors and transporters, followed by their intracellular transport and subsequent recycling or lysosomal/vacuolar degradation (1, 2). In Saccharomyces cerevisiae, transporters usually display both constitutive and accelerated endocytosis regulated by factors such as excess substrate, changes in nutrient availability, and stress conditions. Ubiquitylation of these cell surface proteins acts as a signal triggering their internalization (1). A single essential E3 4 ubiquitin ligase, Rsp5, has been implicated in the internalization of most, if not all, endocytosed proteins (3). Rsp5 is the unique member in S. cerevisiae of the HECT (homologous to E6AP COOH terminus)-ubiquitin ligases of the Nedd4/Rsp5 family (4). In a few cases, Rsp5-dependent cell surface ubiquitylation was shown to involve PY-containing adapters that bind to Rsp5 (5-7). Rsp5-mediated ubiquitylation is also required for sorting into multivesicular bodies (MVBs) of endosomal membrane proteins that come from either the plasma membrane (through endocytosis) or the Golgi (through vacuolar protein sorting (VPS) pathway) (8). Although much progress has been made in elucidating the mechanistic basis of various steps in protein trafficking, the precise requirement for a specific type and length of Ub chains at various stages of the endocytic pathway remains to be addressed.The ubiquitin profile needed for proper internalization has been established for some yeast membrane proteins (1). Th...

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Section: Methodsmentioning

confidence: 99%

Glucose-induced Ubiquitylation and Endocytosis of the Yeast Jen1 Transporter

Paiva

Vieira

Nondier

et al. 2009

Journal of Biological Chemistry

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“…Site-directed mutagenesis of the virB8 genes in was pT7-7StrepIIVirB8sp performed via inverse PCR by using overlapping primers (Table 3, which is published as supporting information on the PNAS web site) that carried the desired mutation (36). Plasmid pTrcP200 was constructed for expression of virB8 genes (WT and mutants) under the control of the trc promoter in a kanamycin resistance-determining vector compatible with pTrcB1ϩ3-12.…”

Section: Methodsmentioning

confidence: 99%

Dimerization and interactions of Brucella suis VirB8 with VirB4 and VirB10 are required for its biological activity

Paschos

Patey²,

Sivanesan³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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VirB8-like proteins are essential components of type IV secretion systems, bacterial virulence factors that mediate the translocation of effector molecules from many bacterial pathogens into eukaryotic cells. Based on cell biological, genetic, and x-ray crystallographic data, VirB8 was proposed to undergo multiple proteinprotein interactions to mediate assembly of the translocation machinery. Here we report the results of a structure-function analysis of the periplasmic domain of VirB8 from the mammalian pathogen Brucella suis, which identifies amino acid residues required for three protein-protein interactions. VirB8 variants changed at residues proposed to be involved in dimerization, and protein-protein interactions were purified and characterized in vitro and in vivo. Changes at M102, Y105, and E214 affected the self-association as measured by analytical ultracentrifugation and gel filtration. The interaction with B. suis VirB10 was reduced by changes at T201, and change at R230 inhibited the interaction with VirB4 in vitro. The in vivo functionality of VirB8 variants was determined by complementation of growth in macrophages by a B. suis virB8 mutant and by using a heterologous assay of type IV secretion system assembly in Agrobacterium tumefaciens. Changes at Y105, T201, R230, and at several other residues impaired the in vivo function of VirB8, suggesting that we have identified interaction sites of relevance in the natural biological context. membrane proteins ͉ type IV secretion ͉ VirB proteins ͉ virulence

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“…For this purpose, oligonucleotide primers designed to cover the sequence encoding each residue to be mutagenized were used in PCR reactions in combination with the overlap extension method (16). The sequence of primers allows the mutagenesis of the sequence Lys-Lys-Arg 63 into Ile-Ile-Ala 63 , that of Lys 32 and Lys 56 into Ile 32 and Ile 56 , respectively, and can be released upon request.…”

Section: Methodsmentioning

confidence: 99%

Site-directed Mutagenesis of the Basic N-terminal Cluster of Pancreatic Bile Salt-dependent Lipase

Aubert¹,

Sbarra

Petit-Thévenin

et al. 2002

Journal of Biological Chemistry

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Previous studies have postulated the presence of a heparin-binding site on the bile salt-dependent lipase (BSDL), whereas two bile salt-binding sites regulate the enzyme activity. One of these sites may overlap with the tentative heparin-binding site at the level of an N-terminal basic cluster consisting of positive residues Lys 32 , Lys 56 , Lys 61 , Lys 62 , and Arg 63 . The present study uses specific site-directed mutagenesis to determine the functional significance of this basic cluster. Mutations in this sequence resulted in recombinant enzymes that were able to bind to immobilized and to cell-associated heparin before moving throughout intestinal cells. Recombinant BSDL was fully active on soluble substrate, but mutants were less active on micellar cholesteryl oleate in comparison with the wild-type enzyme. Activation studies by primary (sodium taurocholate) and by secondary (sodium taurodeoxycholate) bile salts revealed that the activation of BSDL by sodium taurocholate at concentrations below the critical micellar concentration, and not that evoked by micellar bile salts, was affected by substitutions, suggesting that this Nterminal basic cluster likely represents the specific bile salt-binding site of BSDL. Substitutions also affected the activation of the enzyme promoted by anionic phospholipids, extending the function of this site to that of a cationic regulatory site susceptible to accommodate anionic ligands.

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Site-Specific Mutagenesis by Using an Accurate Recombinant Polymerase Chain Reaction Method

Cited by 87 publications

References 2 publications

Glucose-induced Ubiquitylation and Endocytosis of the Yeast Jen1 Transporter

Glucose-induced Ubiquitylation and Endocytosis of the Yeast Jen1 Transporter

Dimerization and interactions of Brucella suis VirB8 with VirB4 and VirB10 are required for its biological activity

Site-directed Mutagenesis of the Basic N-terminal Cluster of Pancreatic Bile Salt-dependent Lipase

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