Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides.
Translocation of lipid-linked oligosaccharide (LLO) intermediates across membranes is an essential but poorly understood process in eukaryotic and bacterial glycosylation pathways. Membrane proteins defined as translocases or flippases are implicated to mediate the translocation reaction. The membrane protein Wzx has been proposed to mediate the translocation across the plasma membrane of lipopolysaccharide (LPS) O antigen subunits, which are assembled on an undecaprenyl pyrophosphate lipid carrier. Similarly, PglK (formerly WlaB) is a Campylobacter jejuni-encoded ABC-type transporter proposed to mediate the translocation of the undecaprenylpyrophosphate-linked heptasaccharide intermediate involved in the recently identified bacterial N-linked protein glycosylation pathway. A combination of genetic and carbohydrate structural analyses defined and characterized flippase activities in the C. jejuni N-linked protein glycosylation and the Escherichia coli LPS O antigen biosynthesis. PglK displayed relaxed substrate specificity with respect to the oligosaccharide structure of the LLO intermediate and complemented a wzx deficiency in E. coli O-antigen biosynthesis. Our experiments provide strong genetic evidence that LLO translocation across membranes can be catalyzed by two distinct proteins that do not share any sequence similarity.
Alaimo
Cristina
C
Department of Biology, Institute of Microbiology, Swiss Federal Institute of Technology, ETH-Hönggerberg, Zurich, Switzerland.
Catrein
Ina
I
Morf
Laura
L
Marolda
Cristina L
CL
Callewaert
Nico
N
Valvano
Miguel A
MA
Feldman
Mario F
MF
Aebi
Markus
M
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016123:Campylobacter jejuni / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D006031:Glycosylation; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D019081:O Antigens / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D026901:Membrane Transport Proteins; D019081:O Antigens; C475086:WzxE protein, E coli; C023023:lipid-linked oligosaccharides; C115291:wlaB protein, Campylobacter jejuni