(E) Negative control for the coupled reaction of PglC/PglA containing Und-[33P]P and UDP-[3H]GalNAc and lacking UDP-diNAcBac

(E) Negative control for the coupled reaction of PglC/PglA containing Und-[33P]P and UDP-[3H]GalNAc and lacking UDP-diNAcBac. polyprenyl groups in the interactions of the glycan substrates, the biosynthetic enzymes that act upon them, and the membrane bilayer in which they are embedded remains a mystery. These interactions are investigated simultaneously and uniquely through application of the nanodisc membrane technology. TheCampylobacter jejuniN-linked glycosylation pathway has been chosen as a model pathway in which all of the enzymes and substrates are biochemically accessible. We present the functional reconstitution of two enzymes responsible for the early membrane-committed steps in glycan assembly. Protein stoichiometry analysis, fluorescence-based approaches, and biochemical activity assays are used to demonstrate the colocalization of the two enzymes in nanodiscs. Isotopic labeling of the substrates reveals that undecaprenyl-phosphate is coincorporated into discs with the two Rabbit polyclonal to DUSP13 enzymes, and furthermore, that both enzymes are functionally reconstituted and can sequentially convert the coembedded undecaprenyl-phosphate into undecaprenyl-diphosphate-linked disaccharide. These studies provide a proof-of-concept demonstrating that the nanodisc model membrane system represents a promising experimental platform for analyzing the multifaceted interactions among the enzymes involved in polyprenol-dependent glycan assembly pathways, the membrane-associated substrates, and the lipid bilayer. The stage is now set for exploration of the roles of the conserved polyprenols in promoting proteinprotein interactions among pathway enzymes and processing of substrates through sequential steps in membrane-associated glycan assembly. Cellular membranes accommodate abundant biological activities, including the transport of small molecules and proteins, energy production, and multistep biosynthetic transformations. These functions are crucial for cell viability; however, studying these processes in biophysical and biochemical detail is challenging because of the complexity of working with both integral and Deferasirox peripheral membrane proteins in lipid bilayer systems. An important class of membrane-associated pathways involves the assembly of complex glycoconjugates, which is dependent on extended linear polyprenols (14). The products of these pathways are essential for cellular viability in all domains of life and, intriguingly, the polyprenols that are used in glycan assembly vary between organisms, with considerable differences in the overall length and degrees of unsaturation (2,5). For example, bacteria generate peptidoglycan components on the membrane-anchored undecaprenyl-diphosphate before cell wall assembly (6), whereas eukaryotes produce glycans for N-linked protein Deferasirox glycosylation via dolichyl-diphosphatelinked intermediates, which feature polyprenols ranging from 1425 isoprene units (1,2,7). Despite the ubiquitous presence of linear polyprenols as glycan carriers in these important biosynthetic pathways, it remains unclear why these structures have been so faithfully conserved throughout evolution and what might be their functional significance, particularly because extended polyprenols have no other known role in cells (2,4,5). The discovery (8) and biochemical investigation (911) of N-linked protein glycosylation in the Gram-negative bacteriumCampylobacter jejunihas revealed a canonical polyprenol-dependent glycan assembly pathway. TheC. jejuniprotein glycosylation (pgl) pathway is an appealing subject for in-depth analysis because Deferasirox the component enzymes can be heterologously expressed and purified in good yields, and can be subjected to protein engineering approaches for the introduction of tags and labels. As illustrated inFig. 1A, in thepglpathway, a heptasaccharide is assembled onto undecaprenyl-diphosphate (Und-P-P) by the sequential action of five membrane proteins, designated as PglC, PglA, PglJ, PglH, and PglI. Of these proteins, PglC and PglI are predicted to be integral membrane proteins (12), whereas the other glycan assembly enzymes (PglA, PglJ, and PglH) lack discrete transmembrane domains (TMDs) and are peripheral membrane proteins. Both the integral and peripheral membrane Pgl proteins form insoluble aggregates in the absence of detergent, which further supports a model wherein these enzymes are recruited to the membrane to collaborate in the sequential glycan assembly process. After biosynthesis of the undecaprenyl-diphosphate heptasaccharide is complete, a flippase (PglK) translocates the assembled product to the periplasmic face of the inner membrane (13), where the oligosaccharyltransferase (PglB) transfers the assembled glycan to asparagine residues in selected acceptor proteins (10,14). After translocation through the outer membrane, glycosylated proteins are displayed on the cellular surface and appear to be involved in adhesion, colonization, and host-cell invasion (15). == Fig. 1. == Canonical reaction pathway of bacterial N-linked glycosylation inC. jejuni. (A) Scheme of the membrane-bound enzymatic conversions that produce the polyprenyl-diphosphatelinked glycan, which is used in protein glycosylation. (B) The reactions of the first two enzymes in theC. jejunipathway, PglC and PglA, are shown. One of the major hurdles in deciphering the discrete biophysical and biochemical roles of polyprenols in membrane-associated pathways is implementing methods to simultaneously investigate all three components involved in polyprenol action: the lipid bilayer, membrane-associated proteins, and polyprenyl-linked substrates. Prior studies on polyprenol-dependent pathways have principally focused on redacted experimental systems.