At the membrane frontier: a prospectus on the remarkable evolutionary conservation of polyprenols and polyprenyl-phosphates

Abstract

Long-chain polyprenols and polyprenyl-phosphates are ubiquitous and essential components of cellular membranes throughout all domains of life. Polyprenyl-phosphates, which include undecaprenyl-phosphate in bacteria and the dolichyl-phosphates in archaea and eukaryotes, serve as specific membrane-bound carriers in glycan biosynthetic pathways responsible for the production of cellular structures such as N-linked protein glycans and bacterial peptidoglycan. Polyprenyl-phosphates are the only form of polyprenols with a biochemically-defined role; however, unmodified or esterified polyprenols often comprise significant percentages of the cellular polyprenol pool. The strong evolutionary conservation of unmodified polyprenols as membrane constituents and polyprenyl-phosphates as preferred glycan carriers in biosynthetic pathways is poorly understood. This review surveys the available research to explore why unmodified polyprenols have been conserved in evolution and why polyprenyl-phosphates are universally and specifically utilized for membrane-bound glycan assembly.

Fig. 1

Structures of fully unsaturated polyprenols and dolichols. The length of the polyprenol and the number of cis and trans isoprene units (m and n, respectively) is species dependent. (Left) Fully unsaturated polyprenols contain all trans isoprene units or a mixture of trans and cis units as shown. (Right) Dolichols are distinguished by a single saturated α-isoprene unit.

Fig. 2

Polyprenol biosynthesis from DMAPP and IPP. Prenyltransferases catalyze the condensation of DMAPP with IPP to form long polyprenyl-diphosphate molecules and direct which stereoisomer (cis or trans) is produced in each reaction. In eukaryotes, the final steps of dolichol biosynthesis involve enzyme-catalyzed reduction of the α-isoprene subunit, which is thought to occur on the unmodified polyprenol, followed by phosphorylation. Specific phosphatases responsible for the hydrolysis of dolichyl-diphosphate have not yet been identified. Bacteria do not require reduction at the α-subunit, and thus their biosynthetic pathways terminate with a single hydrolysis reaction to generate undecaprenyl-phosphate.

Fig. 3. Schematic of N-linked protein glycosylation pathways

(Left) The eukaryotic pathway assembles dolichyl-diphosphate (Dol-PP) tetradecasaccharide (GlcNAc₂Man₉Glc₃) on both the cytoplasmic and luminal faces of the ER membrane prior to glycan transfer. Dol-P-Man and Dol-P-Glc act as glycan donors on the luminal face. (Right) The pathway in the bacterium Campylobacter jejuni assembles an undecaprenyl-diphosphate (Und-PP) heptasaccharide (diNAcBacGalNAc₅Glc) prior to translocation and glycan transfer in the periplasm.

Fig. 4

Dolichyl-phosphate-linked glycan substrates in eukaryotes and archaea. The glycans are depicted in the cellular region where they are biosynthesized and arrows indicate that the glycans undergo protein-catalyzed flipping across the membrane prior to subsequent reactions. (Top) From left to right, the eukaryotic ER membrane contains multiple species, including Dol-P-Man, Dol-P-Glc, Dol-PP-GlcNAc₂Man₅ and Dol-PP-GlcNAc₂Man₉Glc₃. (Lower right) The archaeal membrane contains Dol-P-Man and dolichyl-phosphate-linked glycan precursors to N-linked protein glycans. The glycan produced in Methanococcus voltae is shown, but it is unknown whether this intermediate contains a diphosphate or phosphate linkage, since both have been observed in archaeal species.

Fig. 5

Undecaprenyl-phosphate-linked glycans in bacterial plasma membranes. The glycans are depicted in the cellular region where they are biosynthesized and arrows indicate that the glycans undergo protein-catalyzed flipping across the membrane prior to subsequent reactions. (Top) From left to right, all bacteria contain Und-P-Man and Lipid II, while teichoic acids are only present in Gram-positive bacteria such as the example shown from Staphylococcus aureus. (Bottom) From left to right, representative Und-PP-linked glycans from Gram-negative bacteria are shown including N-linked glycans from C. jejuni, O-linked glycans from N. gonorrhoeae, capsular polysaccharide from E. coli strain K27, heteropolymeric O-antigen subunit from E. coli strain O113, and homopolymeric O-antigen from Klebsiella pneumoniae.

Fig. 6

Activated sugar donors. (Left) Dol-P-Glc is the membrane-bound sugar donor in the ER lumen of eukaryotes. (Right) Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is a soluble sugar donor localized in the cytoplasm.

Fig. 7. Phospholipid structures in presence of polyprenols

(A) Lipids form a typical lipid bilayer in the absence of polyprenol. (B) Some evidence suggests that the hexagonal II conformation (shown here) is induced in the presence of polyprenols. (C) Polyprenol derivatives have different orientations in the membrane. From left to right, a lipid bilayer is shown with polyprenyl-phosphate glycan and polyprenyl-phosphate perpendicular to the membrane, and polyprenol and esterified polyprenol parallel to the membrane surface.

Fig. 8

Proposed structures of polyprenols. From left to right, the structures of undecaprenyl-phosphate, C95 dolichyl-phosphate, and C95 dolichol were determined by NMR and molecular modeling. The red group represents the phosphate. Image reproduced with permission from [110].

Fig. 9

Cellular sources of dolichyl-phosphate. Dolichyl-phosphate has three cellular sources. In the first, dolichol is phosphorylated de novo by a kinase (K) to generate dolichyl-phosphate. In the second and third, a salvage pathway occurs, in which the dolichyl-diphosphate released after glycan transfer is hydrolyzed by a pyrophosphatase (P) or dolicyl-phosphate is released by a glycosyltransferase (G) from Dol-P-sugar donors. In both cases, the dolichyl-phosphate is then translocated from the ER lumen to the cytoplasm by a flippase (F) to begin another round of glycan biosynthesis.

Fig. 10

Proposed structure of PIRS peptide bound to dolichyl-phosphate. Top and side views are shown of dolichyl-phosphate interacting with five PIRS-containing peptides as determined by computational modeling studies and NMR structural data of the peptide (green) and dolichyl-phosphates (yellow). The red, pink and turquoise groups on the peptide denote the two leucines and one isoleucine that contact dolichyl-phosphate. Image reproduced with permission from [109].

Fig. 11

Independent and sequential models of polyprenol-dependent glycan biosynthesis. (A) In the independent model, glycan intermediates are released after every glycosyltransferase reaction. This may result in significant pauses before the intermediate encounters the next enzyme in the pathway. (B) In the sequential model, the enzymes cluster together to produce the glycan efficiently without release of the intermediates to the lipid bilayer.