Construction of phosphatidylethanolamine-less strain of Saccharomyces cerevisiae. Effect on amino acid transport.

A triple yeast mutant was constructed which lacks BST1, the gene for sphingosine lyase, besides the phosphatidylserine decarboxylases PSD1 and PSD2. In this yeast mutant, which can only be grown in the presence of exogenous ethanolamine, phosphatidylethanolamine can be depleted to very low levels. Under those conditions, respiration as well as glucose and 3-O-methylglucose uptake proceed unaffected. Plasma membrane ATPase is as active in these cells as that of control cells grown in the presence of ethanolamine. Drastically decreased, however, are H+/amino acid symporters. The activities of arginine (Can1p), proline (Put4p) and general amino acid permease (Gap1p) are decreased more than 20-fold. Amino acid transport in yeast is dependent on coupling to the proton motive force. It can be envisaged that phosphatidylethanolamine might play a role in this process or in the early steps of the secretion pathway common for all amino acid permeases or, eventually, it could affect the transport proteins directly at the plasma membrane Transformation of the triple mutant with a CEN plasmid harbouring BST1 wild-type gene totally reversed its phenotype to that observed in the double mutant.

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.

The C-terminal tetrapeptide HWFW of the Chlorella HUP1 hexose/H(+)-symporter is essential for full activity and an alpha-helical structure of the C-terminus.

C-terminal tails of plant hexose/H(+)-symporters of the major facilitator superfamily contain a highly conserved motif of four amino acids: HWFW. A deletion of these four amino acids in the Chlorella HUP1 protein leads to a decrease in transport activity by a factor of 3-4. The mutated tail is highly sensitive to trypsin; it does not show alpha-helical conformation in contrast to the wild type C-terminal peptide with an alpha-helical content of at least 15%. The production of monoclonal antibody 416B8 recognizing an epitope within the central loop of HUP1 protein has been a prerequisite for the experiments described.

Expression of eukaryotic plasma membrane transporter HUP1 from Chlorella kessleri in Escherichia coli.

To study the effect of sterols on the activity of the eukaryotic plasma membrane transporter, the hexose-proton symporter HUP1 from the unicellular alga Chlorella kessleri was expressed in Escherichia coli, a prokaryotic microorganism containing virtually no sterols. Under certain conditions, the recombinant protein was partially active in this prokaryotic organism. The heterologously produced HUP1p was purified from membrane fractions of E. coli and reconstituted in an in vitro system. The presence of ergosterol during solubilization, purification and reconstitution resulted in an increased activity of the reconstituted protein. Its activity, however, was 5-6 times lower as compared to the activity of HUP1p produced in Saccharomyces cerevisiae membranes and solubilized, purified, and reconstituted under the same conditions as above.

Purification of the Chlorella HUP1 hexose-proton symporter to homogeneity and its reconstitution in vitro.

A prokaryotic biotin acceptor domain was fused to the carboxy terminal end of the Chlorella hexose-proton symporter. The plant symporter is biotinylated in vivo when expressed in Schizosaccharomyces pombe. The extended biotinylated transport protein is fully active, catalyzes accumulation of D-glucose analogs and restores growth of a glucose-uptake-deficient yeast strain. Crude membranes were solubilized with octyl-beta-D-glucoside in the presence of Escherichia coli L-alpha-phosphatidylethanolamine. Biotinylated symporter was purified to homogeneity by biotinavidin affinity chromatography. The symporter protein was reconstituted together with cytochrome-c oxidase prepared from beef heart mitochondria into proteo-liposomes. Cytochrome-c oxidase is a redox-driven H(+)-pump generating a proton motive force (inside negative and alkaline) while transferring electrons from cytochrome-c to oxygen; this energy is used by the symporter to accumulate D-glucose at least 30-fold. In the absence of the driving force the transport protein facilitates diffusion of D-glucose until the concentration equilibrium is reached. It was shown that maximal transport activity depends highly on the amount of co-reconstituted cytochrome-c oxidase and that the symporter possesses 10% of its in vivo turnover number under optimized in vitro transport conditions.