Heterologous expression of a plant uracil transporter in yeast: improvement of plasma membrane targeting in mutants of the Rsp5p ubiquitin protein ligase.
Plasma membrane proteins involved in transport processes play a crucial role in cell physiology. On account of these properties, these molecules are ideal targets for development of new therapeutic and agronomic agents. However, these proteins are of low abundance, which limits their study. Although yeast seems ideal for expressing heterologous transporters, plasma membrane proteins are often retained in intracellular compartments. We tried to find yeast mutants potentially able to improve functional expression of a whole set of heterologous transporters. We focused on Arabidopsis thaliana ureide transporter 1 (AtUPS1), previously cloned by functional complementation in yeast. Tagged versions of AtUPS1 remain mostly trapped in the endoplasmic reticulum and were able to reach slowly the plasma membrane. In contrast, untagged AtUPS1 is rapidly delivered to plasma membrane, where it remains in stable form. Tagged and untagged versions of AtUPS1 were expressed in cells deficient in the ubiquitin ligase Rsp5p, involved in various stages of the intracellular trafficking of membrane-bound proteins. rsp5 mutants displayed improved steady state amounts of untagged and tagged versions of AtUPS1. rsp5 cells are thus powerful tools to solve the many problems inherent to heterologous expression of membrane proteins in yeast, including ER retention.
Froissard
Marine
M
Institut Jacques Monod-CNRS, Université Paris VI and Paris VII, Paris, France.
Belgareh-Touzé
Naïma
N
Buisson
Nicole
N
Desimone
Marcelo
M
Frommer
Wolf B
WB
Haguenauer-Tsapis
Rosine
R
D029681:Arabidopsis Proteins / Q000096:biosynthesis* / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D016503:Drug Delivery Systems / Q000379:methods; D056827:Endosomal Sorting Complexes Required for Transport; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods*; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics / Q000378:metabolism*
D029681:Arabidopsis Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C460110:ureide permease 1, Arabidopsis; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae