Properties of a reconstituted eukaryotic hexose/proton symporter solubilized by structurally related non-ionic detergents: specific requirement of phosphatidylcholine for permease stability.
Overexpression of the hexose/proton symporter HUP1 from Chlorella kessleri in S. cerevisiae permits a one-step purification via a biotinylation domain. Milligram amounts of the protein are obtained starting from 2 l of yeast culture. The HUP1 protein is used as a model eukaryotic membrane protein of the 'major facilitator superfamily' (MFS) to study specific lipid requirements for activity and stability. Testing two series of detergents revealed that n-nonyl-beta-D-glucoside (NG) and n-octyl-beta-D-glucoside (OG) solubilize the HUP1 protein efficiently. Only the use of NG resulted in long-term stabilization of the HUP1 protein in the absence of external lipids. When affinity purified protein was extracted with organic solvents, a stoichiometric amount of phosphatidyl choline, phosphatidyl ethanolamine and ergosterol in the ratio of close to 2:1 was detected. These lipids were only observed, however, when the protein purification was carried out in the presence of NG; no lipids were copurified with the HUP1 protein in the presence of OG. Of the three lipids copurified, phosphatidyl choline showed a crucial role in ensuring maximal HUP1 permease activity and stability when added back to the OG-protein. The requirement of phosphatidylcholine documents a specific effect of lipids on vectorial transport mediated by a eukaryotic membrane protein of the MFS family.
Robl
I
I
Universität Regensburg, Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universitätsstr. 31, 93053, Regensburg, Germany.
Grassl
R
R
Tanner
W
W
Opekarová
M
M
D019325:3-O-Methylglucose / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002708:Chlorella / Q000378:metabolism*; D003902:Detergents; D004355:Drug Stability; D005947:Glucose / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000494:pharmacology*; D011510:Proteolipids / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D012995:Solubility; D027981:Symporters
D003902:Detergents; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D010713:Phosphatidylcholines; D011510:Proteolipids; D011994:Recombinant Proteins; D027981:Symporters; C089820:monosaccharide-hydrogen cotransporter; C020485:proteoliposomes; D019325:3-O-Methylglucose; D005947:Glucose