Reconstitution of GDP-mannose transport activity with purified Leishmania LPG2 protein in liposomes.
Activated nucleotide sugars required for the synthesis of glycoconjugates within the secretory pathway of eukaryotes are provided by the action of nucleotide sugar transporters (NSTs). Typically, NSTs are studied in microsomal preparations from wild-type or mutant lines; however, in this setting it can be difficult to assess NST properties because of the presence of glycosyltransferases and other interfering activities. Here we have engineered Leishmania donovani to express high levels of an active LPG2 Golgi GDP-Man transporter bearing a C-terminal polyhistidine tag. The functional LPG2-HIS was solubilized, purified by metal affinity chromatography, and reconstituted into phosphatidylcholine-containing liposomes using polystyrene SM-2 beads. The proteoliposomes exhibited robust GDP-Man transport activity with an apparent K(m) of 6.6 mum. Transport activity was enhanced by preloading of GMP and showed specificity for multiple substrates (GDP-Ara and GDP-Fuc). In contrast to the activity in crude microsomes, transport was not dependent on the presence of divalent cations. Thus, reconstitution of transport activity using purified LPG2 protein in liposomes provides firm experimental evidence that a single polypeptide is solely required for NST activity and is able to mediate the uptake of multiple substrates. These studies are relevant to the study of NST structure and function in both protozoan parasites as well as their higher eukaryotic hosts.
Segawa
Hiroaki
H
Department of Molecular and Cellular Biochemistry, University of Kentucky Medical Center, College of Medicine, Lexington, Kentucky 40536, USA.
Soares
Rodrigo P
RP
Kawakita
Masao
M
Beverley
Stephen M
SM
Turco
Salvatore J
SJ
D000818:Animals; D001692:Biological Transport; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006028:Glycosphingolipids / Q000302:isolation & purification / Q000378:metabolism*; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism*; D007893:Leishmania donovani / Q000378:metabolism*; D008081:Liposomes; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015800:Protozoan Proteins / Q000302:isolation & purification / Q000378:metabolism*
D006028:Glycosphingolipids; C095909:LPG2 protein, Leishmania; D008081:Liposomes; D008565:Membrane Proteins; D015800:Protozoan Proteins; D006155:Guanosine Diphosphate Mannose