A novel yeast expression system for the overproduction of quality-controlled membrane proteins.
Saturation of the cell's protein folding capacity and accumulation of inactive incompletely folded protein often accompanying the overexpression of membrane proteins (MPs) presents an obstacle to their efficient purification in a functional form for structural studies. We present a novel strategy for optimization of functional MP expression in Saccharomyces cerevisiae. This approach exploits the unfolded protein response (UPR) pathway, a stress signaling mechanism that senses the accumulation of unfolded proteins in the endoplasmic reticulum. We demonstrate that a high level of UPR induction upon expression of a MP reflects impaired functional expression of that protein. Tuning the expression level of the protein so as to avoid or minimize UPR induction results in its increased functional expression. UPR status can therefore serve as a proxy variable for the extent of impaired expression of a MP that may even be applicable in the absence of knowledge of the protein's biological function.
Griffith
Douglas A
DA
Max-Planck-Institute for Biochemistry, Department of Membrane Biochemistry, Martinsried, Germany. griffith@rzg.mpg.de
Delipala
Christina
C
Leadsham
Jane
J
Jarvis
Simon M
SM
Oesterhelt
Dieter
D
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins; D033703:Nucleoside Transport Proteins; D017510:Protein Folding; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014345:Trypanosoma
D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033703:Nucleoside Transport Proteins; D011994:Recombinant Proteins; C444719:adenosine transporter; D005690:Galactose