At the membrane frontier: A prospectus on the remarkable
evolutionary conservation of polyprenols and polyprenylphosphates
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Hartley, Meredith D., and Barbara Imperiali. “At the Membrane
Frontier: A Prospectus on the Remarkable Evolutionary
Conservation of Polyprenols and Polyprenyl-Phosphates.”
Archives of Biochemistry and Biophysics 517, no. 2 (January
2012): 83–97.
As Published
http://dx.doi.org/10.1016/j.abb.2011.10.018
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Elsevier
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Thu May 26 07:26:26 EDT 2016
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http://hdl.handle.net/1721.1/101254
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At the membrane frontier: A prospectus on the remarkable evolutionary conservation of
polyprenols and polyprenyl-phosphates
Meredith Hartley and Barbara Imperiali*
Department of Biology and Department of Chemistry
Massachusetts Institute of Technology
77 Massachusetts Avenue,
Cambridge, MA 02139
* Phone: (617) 253-1838
Fax: (617) 452-2419
Email: imper@mit.edu.
1
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 function; 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.
Keywords: polyprenol, polyprenyl-phosphate, dolichol, dolichyl-phosphate, N-linked
glycosylation
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Introduction
The long-chain polyisoprenoid alcohols (polyprenols and dolichols) are a unique class of
secondary metabolites within the isoprenoid natural product family (Fig. 1). These
polyisoprenols comprise a small percentage (0.1%) of the total phospholipids in cellular
membranes of both prokaryotes and eukaryotes [1-3]. Polyprenyl-phosphates act as
oligosaccharide carriers during glycan biosynthesis, which is essential to many conserved
cellular processes including N-linked protein glycosylation, C- and O-protein mannosylation,
and bacterial cell wall biosynthesis. Despite extensive research on the enzymes that utilize
polyprenols in glycan assembly pathways, relatively little is known about why polyprenols
prevail as the most common glycan carriers in nature. This review begins with an overview of
polyprenol structure and polyprenyl-phosphate dependent processes in biology. After providing
essential background on the “what” and “where” of polyprenols, we will address the more
interesting question of “why polyprenols in the first place?”
Structural features of long-chain polyisoprenols
The linear polyprenols (or polyisoprenols) contain 7 to 24 isoprene units in either the
trans or cis configuration and can broadly be separated into two subclasses [3-6]. The first class
contains only unsaturated isoprene units such as undecaprenol and related homologs, whereas the
second class is distinguished by the presence of a single saturated isoprene unit in the α-position
(Fig. 1). In both classes, α refers to the unit closest to the hydroxyl moiety and ω is used to
designate the terminal isoprene unit in the linear structure. Very recently, a third class of linear
polyprenols termed „alloprenols‟ has been identified in several plant species. Alloprenols possess
a similar structure to the first class of polyprenols, but contain a trans isoprene unit at the αposition [7, 8].
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Many studies have focused on elucidating the process of polyprenol biosynthesis, which
involves the enzyme-catalyzed condensation of dimethylallylpyrophosphate (DMAPP)1 with
multiple units of isoprenylpyrophosphate (IPP) (Fig. 2). The biosynthesis of isoprenoid alcohols
has been reviewed recently [5, 6, 9-11]. In general, the polyprenols featured in glycan assembly
pathways vary in length across organisms, ranging from C55 polyprenols in bacteria and C95
dolichols in mammals to C200 polyprenols in plants (Table 1). Bacteria typically exploit
undecaprenol, a C55 unsaturated polyprenol with the ω isoprene unit followed by two trans
isoprene units and eight cis isoprene units [12]. Archaeal organisms typically contain C55-C66
dolichols [13], Saccharomyces cerevisiae (yeast) contain C80 dolichols [14] and mammalian
tissues contain C90-C100 dolichols (Table 1) [15]. In addition, unusual polyprenols that include
additional saturated isoprene units at the ω-terminus have been identified in Mycobacterium [16,
17] as well as the archaeal species, Haloferex volcanii [18] and Sulfolobus acidocaldarious [19].
Plants exhibit the widest range of polyprenol diversity, as both unsaturated polyprenols
and dolichols with a wide range of lengths have been characterized [20-22]. The lipid bilayers of
most plants contain C55 polyprenols, although in contrast to undecaprenol, these polyprenols
contain three, rather than two, trans isoprene units, due to small differences at the beginning of
the biosynthetic pathway [9]. Plants in the gymnosperm family contain C80-C100 polyprenols
[21], while plants in the genus Potentilla contain mixtures of polyprenols with the longest
possessing up to 200 carbons [23]. It is interesting to note the wide diversity of linear polyprenol
1
Abbreviations used: CHO, Chinese hamster ovary; Dol-P, dolichyl-phosphate; dolichylphosphate mannose; Dol-PP, dolichyl-diphosphate; DMAP, dimethylallylpyrophosphate; ER,
endoplasmic reticulum; GalNAc, N-acetylgalactosamine; Glc, glucose; IPP,
isopentylpyrophosphate; Man, mannose; MurNAc, N-acetylmuramic acid; NDP, nucleotide
diphosphate; UDP, uridine diphosphate; Und-P, undecaprenyl-phosphate; Und-PP,
undecaprenyl-diphosphate.
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architectures that has evolved amongst the three kingdoms of life, as the known functions of
these molecules are very similar in all organisms.
Polyprenyl-phosphate as glycan carriers in biosynthetic pathways
Polyprenyl-phosphates are essential substrates for critical cellular functions in both
eukaryotes and prokaryotes. These roles include N- and O-linked protein glycosylation in
eukaryotes, archaea and bacteria and the biosynthesis of central structural components in bacteria
such as peptidoglycan and O-antigen. Of these biosynthetic processes, N-linked protein
glycosylation may be the most well known and the role of polyprenols in this process has been
recently reviewed by Krag and coworkers [6]. It involves the stepwise assembly of an
oligosaccharide on polyprenyl-diphosphate or polyprenyl-phosphate and occurs in eukaryotes,
archaea, and several bacterial species (Fig. 3) [6, 24, 25]. In eukaryotes, a conserved dolichyldiphosphate (Dol-PP) heptasaccharide is assembled on the cytoplasmic face of the endoplasmic
reticulum (ER) membrane (Fig. 4) [26-28]. The Dol-PP-glycan intermediate is then translocated
across the lipid bilayer to the ER lumen, where it is further glycosylated to form a
tetradecasaccharide that is transferred to the nitrogen in the primary amide side chain of
asparagine residues in nascent proteins (Fig. 4).
To date, characterization of N-linked glycosylation in archaea is limited, but these
pathways are known to involve assembly of small, highly modified glycans on dolichylphosphate (Dol-P) carriers, such as the glycan depicted in Fig. 4 from Methanococcus voltae [29,
30]. Dolichyl-phosphate, as opposed to dolichyl-diphosphate, may act as the glycan carrier in
certain archaeal species. Glycans linked to dolichol via mono- and diphosphate bridges have
been identified [13, 18, 31-33], but Dol-P-glycans are used exclusively by archaea such as
Haloarcula marismortui [34] and Haloferax volcanii [18]. It is intriguing that some archaeal
5
species have evolved to use a monophosphate linkage exclusively in glycan assembly for Nlinked glycosylation, and it may have to do with the greater stability of Dol-P-glycans. For
instance, the diphosphate linkage is susceptible to hydrolysis under milder conditions than the
monophosphate [13]. In addition, the β-glycosidic linkages of Dol-P-glycans, as opposed to the
α-glycosidic linkages in Dol-PP-glycans, endow these molecules with greater stability, since the
lone pair of electrons on oxyen is not positioned to facilitate the C-O bond cleavage. Although
the stereochemistry of Dol-P-glycans in archaea has not yet been determined, Dol-P-glycans
found elsewhere in nature contain a β-glycosidic linkage. If monophosphate β-linkages are
common in archaeal species, it could represent a way that these organisms have adapted to the
extreme conditions of archael habitats.
Over ten years ago, the first bacterial N-linked glycosylation pathway was identified in
the Gram-negative pathogen Campylobacter jejuni [35] and was later validated by the transfer of
the operon into Escherchia coli [36]. More recently, genomic analysis suggests that homologous
N-linked glycosylation pathways may be found in at least 84 different prokaryotic species [37].
The C. jejuni pathway represents the most well-characterized bacterial glycosylation system [25,
38-44], and involves the biosynthesis of an undecaprenyl-diphosphate (Und-PP) heptasaccharide
prior to glycan transfer to asparagine in proteins in the periplasmic space (Figs. 3 and 5). The
similarities of the C. jejuni N-linked pathway to the eukaryotic pathway have been recently
reviewed [24, 25], but it is important for the following discussion to note that both pathways rely
on the stepwise addition of glycans to a membrane-anchored polyprenyl-phosphate carrier (Fig.
3). An interesting side note to bacterial N-linked glycosylation is that a polyprenyl-phosphate
dependent O-linked protein glycosylation pathway has been discovered in Neisseria species [4548]. O-linked glycosylation is a ubiquitous process in eukaryotes and prokaryotes, but this is the
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first example in which a glycan destined for a serine or threonine residue is assembled on
undecaprenyl-diphosphate (Fig. 5) prior to en bloc transfer to protein [45, 46, 49, 50] rather than
sequential transfer of monosaccharide units to assemble the glycan on the protein.
In addition to the role in bacterial protein glycosylation, undecaprenyl-phosphate acts as a
membrane-associated carrier in the biosynthesis of many extracellular, oligosaccharide-based
structures in bacteria (Fig. 5). Importantly, peptidoglycan intermediates are biosynthesized on an
undecaprenyl-phosphate carrier; these intermediates include Und-PP-MurNAc(pentapeptide)
(Lipid I) and Und-PP-MurNAc(pentapeptide)-GlcNAc (Lipid II). After Lipid II is translocated
across the membrane, peptidoglycan is assembled through disaccharide polymerization and
peptide cross-linking forming a rigid cell wall barrier that is essential for bacterial viability.
Because peptidoglycan biosynthesis has provided excellent targets for antibiotic development,
much effort has focused on understanding how Lipid II precursors are biosynthesized and
incorporated into the cell wall as described in the following references [10, 51, 52]. Several other
essential bacterial components are synthesized via undecaprenyl-diphosphate intermediates
including O-antigen polymers [53, 54], teichoic acids [55-58], and capsular polysaccharides [5961]. These extracellular glycans are involved in bacterial defense mechanisms, mediate crucial
microbial-host interactions and may also represent potential antibacterial target pathways.
Polyprenyl-phosphates as glycan donors
In addition to acting as glycan carriers, polyprenyl-phosphates play a second, essential
biosynthetic role by acting as activated glycan donors for transfer to protein or other biosynthetic
intermediates; these molecules are membrane-bound alternatives [62] to nucleotide-diphosphate
(NDP) activated donors (Fig. 6). Polyprenyl-phosphate monosaccharides are assembled on the
cytoplasmic surface of a cellular membrane from an NDP-sugar and polyprenyl-phosphate; these
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substrates are then translocated across the ER membrane in eukaryotes or the plasma membrane
in prokaryotes. Once translocated, they serve as glycan donors in the ER lumen or periplasm,
where NDP-sugars are absent (Figs. 4 and 5). In this way, cellular systems exploit polyprenyl
translocation to transport activated sugar donors to regions that require glycan biosynthesis, but
lack NDP-sugars.
Specifically, dolichyl-phosphate mannose (Dol-P-Man) and dolichyl-phosphate glucose
(Dol-P-Glc) act as membrane-bound monosaccharide donors for a variety of biosynthetic
purposes (Fig. 4). Importantly, Dol-P-Glc and Dol-P-Man participate in eukaryotic N-linked
glycosylation by serving as monosaccharide donors for the assembly of the Dol-PPtetradecasaccharide on the ER luminal interface [1]. Dol-P-Man is also the mannose donor for
glycosylphosphatidylinositol (GPI)-anchor biosynthesis in the ER lumen, which is an essential
process responsible for attaching eukaryotic proteins to the plasma membrane [63]. Dol-P-Man
can also serve as a glycan donor for direct glycosylation of protein substrates. For instance,
protein O-mannosylation relies on direct transfer of mannose from a Dol-P-Man donor in
eukaryotes and archaea, whereas Und-P-Man is the donor in bacteria [62]. In addition, Cmannosylation of tryptophan residues in proteins via Dol-P-Man is a rare modification that has
been identified in a handful of eukaryotic proteins [64].
Evolutionary conservation of polyprenols and polyprenyl-phosphates
In contrast to the well-established responsibilities of polyprenyl-phosphates in biology,
free polyisoprenols have no clearly identified functions in biological systems, despite the fact
that they appear to accumulate at high levels within certain organisms [1, 15, 21, 65-69]. As a
result of this observation, it is believed that these compounds may play a distinct role in cellular
physiology, rather than simply providing a substrate pool for polyprenyl-phosphate dependent
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processes. In the remainder of this review, the evolutionary conservation of polyprenols and
polyprenyl-phosphates will be examined. In the next section we explore the potential roles of
unmodified polyprenols in light of the distribution and abundance of polyprenols in eukaryotic
and bacterial membranes. Then we will evaluate evidence for three hypotheses to explain why
polyprenyl-phosphates may be the preferred glycan carriers in biology. (1) Polyprenylphosphates exert regulatory roles controlling glycan biosynthesis and ultimately cell growth. (2)
Glycan translocation across lipid bilayers is a ubiquitous feature of all polyprenyl-phosphatedependent pathways, suggesting that polyprenol structures may be involved in the enzymemediated mechanism. (3) Glycan assembly pathways reliant on polyprenol derivatives often
contain many membrane-associated enzymes, which potentially form macromolecular
complexes at the lipid bilayer interface dependent on the presence of polyprenyl-phosphate
substrates.
Why polyprenols?
Role of dolichols in eukaryotes
Although polyprenyl-phosphates have been identified in many biochemical processes,
unmodified polyprenols and esterified polyprenols are also present in biological membranes, and
the function of these molecules has not been well defined [1, 3, 9, 70, 71]. The concentrations of
dolichol and dolichyl-phosphate have been determined for a variety of eukaryotic cell types
including mammalian cell culture lines and tissue extracts from rats and humans using several
quantification techniques (Table 2). These studies establish that a significant portion of the
dolichol pool is in the unmodified alcohol form.
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Close examination of the values in Table 2 reveals discrepancies in both the absolute and
relative amounts of dolichol and dolichyl-phosphate. Some inconsistency is likely due to
differences in the extraction, purification and quantification protocols. A recent review highlights
the latest liquid chromatography/tandem mass spectrometry methods used for polyprenol
isolation [72]. In general, Table 2 indicates that unmodified polyprenols constitute from as little
as 9% to as much as 90% of the polyprenol pool depending on the organism and tissue type. One
study suggests polyprenols tend to accumulate over the lifetime of the organism in a variety of
tissues types [67], which is a phenomenon that has been observed in plants as well [21, 73] and
may also account for other inconsistencies observed in Table 2. Importantly, further examination
of the data suggests that the amount of unmodified dolichol increases with age, whereas
dolichyl-phosphate levels remain constant [67]. This finding has also been verified in human
brain tissue, where free dolichols appear to accumulate (3 to 20-fold) over a lifespan, but
dolichyl-phosphate levels remain relatively constant [74-76]. The cause of this increase is
unknown, but it is suggestive that the accumulation of polyprenols such as dolichol could be a
factor in the side effects of aging [9, 71].
In addition to the potential role in aging, altered free dolichol levels have been measured
in a variety of disease states suggesting that an increased dolichol concentration could serve as a
biomarker for various diseases [9, 71] including liver cancer and cirrhosis, cataract disease,
lysosomal storage disorders, and neurodegenerative diseases including Alzheimer‟s and neuronal
ceroid lipofuscinosis [15, 71, 77-82]. Several studies in plants and mammals have indicated that
polyisoprenols may have antioxidant properties and participate in scavenging of free radical
oxygen species [83-85]. If this result is further validated, then it could provide some explanation
for the elevated dolichols levels in disease states [9].
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Studies on the cellular localization of dolichols have also suggested alternative functions
for native and esterified dolichols. As expected, the endoplasmic reticulum membrane, which is
the site of N-linked glycosylation, contains a significant amount of dolichol [70]. More
surprisingly, several studies identified lysosomes as having high levels of dolichol derivatives
that are esterified with a variety of fatty acids [70, 86]. This observation is interesting in light of
the finding that altered dolichol levels were observed in lysosomal storage disorders. It has been
suggested that dolichols could play a role in the intracellular trafficking of fatty acids, or
alternatively, that fatty acid derivatives of dolichols act as transport vehicles for dolichols [70].
In addition, a key enzyme in dolichol biosynthesis, the cis-prenyltransferase, has been implicated
in protein trafficking and retention within the dynamic ER and Golgi membrane network,
suggesting that dolichol and its derivatives may a play a role in protein localization [87, 88].
Role of undecaprenol in bacteria
Most studies on polyprenols have focused on mammalian tissues and plants, as it is more
facile to extract significant amounts of material from these macroscopic sources. However, the
bacterial polyprenol, undecaprenol (also termed „bactoprenol‟) is a key mediator in the
biosynthesis of many essential bacterial structures including peptidoglycan, and a few studies
have examined the relative populations of various undecaprenyl derivatives in bacterial
membranes (Table 3). Interestingly, the Gram-negative bacterium, Escherichia coli, was found
to contain only undecaprenyl-phosphate and undecaprenyl-diphosphate with no reserve pool of
free undecaprenol [89]. In contrast, several studies revealed significant amounts of free
undecaprenol in Staphylococcus aureus and other Gram-positive bacteria like Streptococcus
faecalis (Table 3) [90]. It should be noted that no undecaprenol kinases have been identified in
Gram-negative bacteria, whereas several have been identified in Gram-positive bacteria [91-94].
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In contrast, undecaprenyl-diphosphate phosphatases have been identified in both Gram-negative
and Gram-positive bacterial species [10, 59, 95-97] and have been shown to be important both
for the regeneration of undecaprenyl-phosphate from undecaprenyl-diphosphate in E. coli [98].
Taken together, this evidence may suggest that E. coli do not require unmodified undecaprenol,
because undecaprenyl-phosphate is primarily produced from undecaprenyl-diphosphate via the
action of a phosphatase. Further studies are imperative to determine if low levels of
undecaprenol is a shared characteristic of all Gram-negative bacteria.
Biophysical effects of polyisoprenols on membranes
The potential roles of polyprenols and dolichols in membranes have also been examined
with biophysical approaches focused on elucidating the effects of these molecules in model
membrane vesicles. A variety of techniques have been used to address this question including
nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry, small angle
X-ray diffraction, electrophysiology, and freeze-fracture electron microscopy [99-108]. From
these studies, there is considerable evidence that polyisoprenols increase membrane fluidity, ion
permeability and the propensity of membranes to adopt a non-bilayer, hexagonal II conformation
(Fig. 7). Interestingly, a recent study determined that alloprenols, a newly discovered class of
polyprenols in plants containing an α-isoprene unit in the trans configuration, increase
membrane permeability to a greater extent than the more common cis configuration [4, 7]. The
authors postulated that trans/cis isomerization may be one way that plant cells regulate
membrane permeability [4].
Additional structural information is gleaned from these experiments. Studies have
suggested that dolichol and dolichyl esters are oriented parallel to the plane of the membrane and
12
form aggregates near the center of the bilayer, whereas dolichyl-phosphates are monodispersed
and oriented perpendicular to the membrane with the charged headgroup at the hydrophilic
surface (Fig. 7) [101, 104]. In addition, small-angle X-ray scattering and NMR studies [102, 107109] suggest that the long-chain polyprenols (Table 1) do not adopt a linear conformation and
instead form three separate domains with a coiled center region such that they can easily reside
within the width of the lipid bilayer.
One caveat with many of the reported studies is that biophysical experiments on
polyisoprenols are performed in model membranes containing pure phospholipids. These model
membranes may be inadequate substitutes for native phospholipid bilayers, which contain a
diverse array of phospholipids and membrane-associated proteins. Indeed, in a recent study, the
inclusion of a small hydrophobic peptide into dolichol-containing membrane vesicles reversed
the bilayer destabilization caused by dolichols [107, 108]. Clearly, further studies are needed to
understand the effects of polyisoprenols in the context of physiological membranes, but it is
apparent from the biophysical studies that have been reported to date that dolichol and dolichylphosphate have a profound effect on membrane properties. In the future it will be interesting to
understand whether the biophysical effects of dolichol accumulation in the phospholipid bilayer
could contribute to aging or other cellular processes. In addition, these biophysical studies may
suggest that the absence of undecaprenol in E. coli has a structural role in maintaining the unique
dual membrane architecture of Gram-negative bacteria, as it would minimize membrane
fluidization. However, the undecaprenol levels have not been examined in other Gram-negative
bacteria, and more work is needed in this area. In both eukaryotes and prokaryotes, much
remains to be understood about the presence or absence of unmodified polyprenols, however, the
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quantification and biophysical studies presented here are important first steps towards
understanding how these molecules may be exerting their influence on biology.
Why polyprenyl-phosphates? Regulation of polyprenyl-phosphate dependent pathways
Although our understanding of the roles and functions of unmodified polyprenols is still
at an early stage, significant research has focused understanding the ways in which polyprenylphosphates are utilized at a cellular level. In this section, the role of polyprenyl-phosphates as
regulators of essential cellular processes will be explored as a potential explanation for why
polyprenyl-phosphates are ubiquitous glycan carriers in both eukaryotes and prokaryotes. The
following two hypotheses, which propose a putative role for polyprenols in glycan translocation
and multi-enzyme complex formation, are highly speculative, but the role of polyprenylphosphates as regulatory molecules is better understood and the current status of that research
will be summarized here.
Dolichyl-phosphate as a regulatory molecule in eukaryotes
The importance of dolichyl-phosphate in eukaryotes is well established [3, 11, 110]
[71]and was first demonstrated in several early studies, which suggested that the absolute amount
of dolichyl-phosphate modulated the levels of glycoprotein biosynthesis in eukaryotes [2, 111114]. In one study, estrogen-induced differentiation of hen oviducts resulted in increased
production of Dol-P-[14C]Man when incubated with GDP-[14C]Man [112]. Further
experimentation determined that the elevated amounts of Dol-P-[14C]Man could be attributed to
higher levels of dolichyl-phosphate and not to increased enzymatic activity, thus implying that
increased production of dolichyl-phosphate is an important regulator of cell differentiation [112].
In a related study, incubation of sea urchin embryos with an inhibitor of polyisoprenol
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biosynthesis resulted in abnormal gastrulation, which was correlated with the inability of the cell
to produce glycoproteins. Furthermore, addition of exogenous dolichol allowed for normal
gastrulation, suggesting that dolichyl-phosphate is a limiting reagent for N-linked glycoprotein
biosynthesis and subsequent cellular transformations [115]. The rate of dolichyl-phosphate and
glycoprotein synthesis has also been linked to the growth rate of CHO cells and cell division [2].
Other studies have confirmed that dolichyl-phosphate is a rate limiting substrate in N-linked
glycosylation and is thereby a key factor in cellular development [1, 111]. It is clear from these
studies that dolichyl-phosphate levels regulate the amount of glycoprotein biosynthesis, thus
affecting important transitions during cellular development and growth . Because dolichol
biosynthesis operates independently from phospholipid biosynthesis, it can be differentially
regulated such that production of glycoproteins is unrelated to other membrane-bound processes.
If the amount of dolichyl-phosphate controls the rate of cellular development, then
dolichyl-phosphate biosynthesis must also be regulated during growth. The biosynthesis of
dolichyl-phosphate, outlined in Fig. 2, is a complex process that could potentially be regulated at
every step. A recent study has identified the Nogo-B receptor as important for regulation of the
cis-isoprenyltransferase, which is responsible for the final polyprenol elongation with cis
isoprene units [116]. Nogo-B receptor was found to be essential for the stability of the enzyme
and provides some of the first experimental evidence into how dolichol biosynthesis might be
regulated [116]. It will be important to learn how this regulation correlates to the requirement for
dolichyl-phosphate in developing cells.
Dolichyl-phosphate is metabolically generated in two ways; it can be synthesized de novo
by phosphorylation of dolichol or regenerated by hydrolysis of dolichyl-diphosphate (Fig. 8). For
the first route, a CTP-dependent dolichol kinase has been identified, which has been shown to be
15
important during cellular development [117]. However, de novo phosphorylation of dolichol is
not considered to be the most important source of dolichyl-phosphate (Fig. 8). Instead, dolichylphosphate is regenerated through the action of a pyrophosphatase that hydrolyzes dolichyldiphosphate, which is the byproduct after glycan transfer reactions in the N-linked protein
glycosylation pathway. The regenerated dolichyl-phosphate is then flipped by an unknown
protein catalyst to the cytoplasmic face of the ER membrane to begin another cycle of glycan
assembly. A recent study has established that this second path is the principal source of dolichylphosphate in ER membranes (Fig. 8) [115, 118]. This suggests that while production of dolichylphosphate by de novo phosphorylation of dolichol may be important during periods of rapid
cellular growth, the recycling of dolichyl-diphosphate by a pyrophosphatase may play a more
significant regulatory role in the maintenance of dolichyl-phosphate levels in non-dividing cells.
Another crucial step in dolichol biosynthesis is reduction of the α-isoprene unit by
polyprenol reductase [69]. This key step involves the conversion of the polyprenol into dolichol
(Fig. 2) and occurs after the long-chain polyprenol has been assembled from IPP and DMAPP. It
is believed that phosphatases convert the polyprenyl-diphosphate to polyprenol prior to the
reduction step, which acts as an additional point of regulation for glycoprotein production.
The essentiality of the enzymes involved in dolichyl-phosphate production is underscored
by the class of diseases known as congenital disorders of glycosylation (CDGs) [119-121].
CDGs are characterized by aberrant glycosylation, which can arise from mutations in the
biosynthesis of both dolichols [122] and N- and O-linked glycoproteins [123], and to date 45
different CDGs have been identified [119]. Originally these diseases were divided into two
classes based on the origin of the glycosylation defect; disorders of CDG type I resulted from
mutations in genes encoding for the production of Dol-PP-GlcNAc2Man9Glc3 (Figure 4),
16
whereas CDG type II contained defects in genes whose products are involved in glycan
processing in the Golgi. However, due to the increasing numbers of identified diseases, recently
a new nomenclature was proposed, which utilizes the name of the defective gene to identify the
particular disorder [120]. Importantly, CDGs have been identified that are related to mutations in
dolichol kinase and polyprenyl-diphosphate reductase (Fig. 8) [123, 124], two enzymes that are
responsible for production of dolichyl-phosphate. From the standpoint of CDGs, it is imperative
to understand the regulation of dolichyl-phosphate in order to determine how best to treat
patients with defects in dolichyl-phosphate biosynthesis.
Role of undecaprenyl-phosphate and Lipid I/II in bacteria
In bacteria, undecaprenyl-phosphate serves as the glycan assembly carrier of choice,
however, there is little evidence that it plays a regulatory role similar to that of dolichylphosphate. Limiting amounts of undecaprenyl-phosphate or Lipid II, the undecaprenyldiphosphate linked monomer of peptidoglycan, (Fig. 5) could substantially decrease cell wall
production and thereby cellular growth. However, as described in the previous section, only a
few studies have examined the amount of undecaprenol derivatives in bacteria (Table 3).
Undecaprenyl-phosphate appears to comprise a substantial portion of the available polyprenol
pool in both E. coli and S. aureus suggesting that it may not be a rate-limiting substrate under
these conditions [89]. However, the undecaprenol kinase from Streptococcus mutans has been
identified as a virulence factor, which suggests that undecaprenyl-phosphate production may
play a role in bacterial adaptation to stress [92]. Further studies are needed to discern if
production of undecaprenyl-phosphate is an important regulator in bacteria.
Only a handful of studies have attempted to quantify the amount of Lipid II in bacteria,
which could also regulate cell wall biosynthesis. Metabolic labeling has been used in E. coli to
17
quantify the pools of Lipid I/II relative to other substrates required for peptidoglycan
biosynthesis resulting in estimation of ~3000 copies per cell [125]. Based on the work described
in Table 3 [89], this would represent <2% of the undecaprenyl-phosphate supply in E. coli. It is
important to note that the amount of Lipid I/II in E. coli has not been measured directly, and
further quantitative studies could provide greater insight [125]. In contrast, the amount of Lipid
I/II has been estimated in a variety of Gram-positive bacteria and in every case the amount is
much higher than in E. coli. Metabolic labeling was used to determine that each cell of Bacillus
megaterium contained approximately 34,000 Lipid I/II molecules [126]. Other studies estimated
the amount by determining the number of binding sites for various Lipid II targeting antibiotics
such as ramoplanin and mersacidin [127]. These reports suggest that there are 50,000
peptidoglycan precursors in S. aureus [128], 70,000 in Micrococcus luteus [129], and 200,000 in
Staphylococcus simulans [129]. In S. aureus, this amount of Lipid I/II would represent over 40%
of the total pool of undecaprenyl derivatives, a considerably larger percentage than in E. coli.
Similar to the studies on undecaprenyl-phosphate, further work is needed to more
accurately quantify the amount of Lipid I/II present in a wide range of bacterial species.
However, from the available data, it appears that E. coli and S. aureus contain significantly
different levels of undecaprenol and Lipid I/II. Since Gram-positive bacteria have much thicker
peptidoglycan layers (20-80 nm) than Gram-negative (7-8 nm), it is not surprising that Grampositive bacteria may require larger pools of Lipid I/II. In addition, the differences may imply
that Gram-negative and Gram-positive bacteria rely on different mechanisms for the regulation
of cell wall biosynthesis and other undecaprenyl-phosphate dependent processes. Further studies
are needed to determine how Gram-negative and Gram-positive bacteria regulate the production
18
of undecaprenyl-phosphate and Lipid I/II and if this represents a general physiological
distinction.
Polyprenyl-phosphates are shared amongst competing enzymes and pathways
An interesting facet of polyisoprenol regulation is that in most cases these molecules are
substrates for several competing pathways or enzymes. In eukaryotic systems, three
glycosyltransferases utilize dolichyl-phosphate to produce the three dolichyl substrates required
for N-linked glycosylation: Dol-P-Man, Dol-P-Glc and Dol-PP-N-acetylglucosamine (Dol-PPGlcNAc) [1]. It has been shown that the activities of these three transformations are linked in
vivo such that if one enzyme is inhibited, the activities of the other two increase with the
availability of dolichyl-phosphate substrate [1]. These results imply that the enzymes share a
common substrate pool and further confirm that dolichyl-phosphate concentrations can limit
production of glycoproteins.
In eukaryotes, the three reactions requiring dolichyl-phosphate are all involved in Nlinked glycosylation, whereas in bacteria, enzymes require undecaprenyl-phosphate for separate
biosynthetic ends. For instance, Bacillus licheniformis contains both peptidoglycan and teichoic
acids built on undecaprenyl-phosphate. In one study, it was found that inhibition of
peptidoglycan biosynthesis by omission of the glycan donor or by treatment with the antibiotic
bacitracin did not lead to significant decreases in the production of teichoic acids, suggesting that
the two pathways are not limited by a common pool of undecaprenyl carrier [130]. This led the
authors to posit the presence of multi-enzyme complexes capable of internally recycling
undecaprenyl-phosphate after each biosynthetic cycle, rather than releasing it into the substrate
pool after each round of synthesis [130]. While these findings only represent one study, it is clear
that undecaprenyl-phosphate consumption in bacteria is complicated by the presence of several
19
biosynthetic routes. Further investigations are required to understand how bacteria regulate the
availability of undecaprenyl-phosphate to the various pathways in which it is implicated.
Enzyme specificity for polyprenols
Most eukaryotic enzymes that rely on dolichol derivatives as substrates are specific for
dolichol [131-136] and typically prefer S(-) dolichol to either R(+) dolichol or the corresponding
unsaturated polyprenol of a similar length indicating specificity for the configuration of the αisoprene subunit [131]. Correspondingly, several studies have indicated that bacterial several
polyprenol-binding enzymes are specific for undecaprenol, and many of these are summarized in
the following reference [137]. A recent study examined three glycosyltransferases in the
bacterial N-linked glycosylation pathway in C. jejuni and found that all were specific for both the
degree of saturation and cis/trans configurations of the isoprene units [40]. Interestingly, this was
observed even in the case of a soluble glycosyltransferase that is not predicted to contain any
transmembrane domains. Enzyme specificity for polyisoprenols in prokaryotes and eukaryotes is
an important component of polyisoprenol regulation because it allows these molecules to
specifically interact with cognate enzymes independently in the milieu of the phospholipid
bilayer.
Why polyprenyl-phosphates? Importance in glycan “flipping”
Polyprenyl-phosphate dependent pathways in bacteria, archaea, and eukaryotes rely
universally on translocation or “flipping” of polyprenol derivatives across lipid bilayers [118,
133, 138-143]. However, very little is known about the active roles of proteins and the
polyprenol moieties of the substrates in the mechanism of translocation. Studies using a variety
of biophysical and biochemical methods have established that polyprenyl-phosphates and
20
glycan-modified polyprenols have very slow trans-bilayer diffusion rates (t1/2 = >2 h) in model
membranes when unassisted by protein catalysis [105, 106, 144]. Biosynthetic rates for the
production of N-linked glycans and peptidoglycan are such that relatively fast rates of glycan
translocation are required in both eukaryotes and prokaryotes (t1/2 = <1 s) [125, 145].
Furthermore, in vivo studies have implied that the majority of dolichyl-phosphate is regenerated
through a recycling mechanism reliant on translocation (Fig. 8) [118], which would also require
much faster rates than the uncatalyzed diffusion models. The difference between the observed
and required flipping rates implies that catalysis has led the long-standing hypothesis that
proteins known as flippases catalyze the translocation of polyprenyl-phosphate derivatives. This
section will summarize our current understanding of flippases with a description of the proposed
protein-based and polyprenol-based mechanisms for the translocation reaction.
Eukaryotic and bacterial flippases
A variety of dolichyl-phosphate derivatives, including Dol-PP-GlcNAc2-Man5, Dol-PMan, Dol-P-Glc, and Dol-P, are flipped across the ER membrane to facilitate N-linked
glycosylation and related processes (Fig. 4) [145]. However, until the last decade, relatively the
proteins involved in the process had not been identified or characterized biochemically. The
development of in vitro flippase assays has allowed for the isolation of distinct protein-based
activities for the translocation of Dol-PP-GlcNAc2-Man5 [142, 146, 147] and Dol-P-Man [140],
and research is ongoing to identify the protein catalysts responsible for the observed flippase
functionalities.
Similar to eukaryotic flippases, it is thought bacteria contain different flippases that
catalyze the translocation of the unique undecaprenyl-phosphate-linked substrates from each
pathway. The genes encoding bacterial pathway enzymes are frequently found in clustered
21
operons, which have enabled relatively facile identification of flippases, in contrast to the
identification of eukaryotic flippases. Interestingly, it has been shown that two distinct protein
families perform this function in prokaryotes [138]. The first class of flippases is the ABC-type
transporters, which have an associated ATPase activity and are implicated in N- and O-linked
protein glycosylation as well as the biosynthesis of homopolymeric O-antigen, which is
polymerized in the cytoplasm before it is flipped to the periplasm (Fig. 5). The second class of
flippases, wzx-type translocases, shares homology with eukaryotic flippases and shows no ATP
dependence. Wzx-flippases are responsible for translocation of the individual subunits of
peptidoglycan, heteropolymeric O-antigen, capsular polysaccharides and teichoic acids across
the plasma membrane (Fig. 5), which is followed by polymerization in the periplasm. It has been
proposed that polymerization may be a driving force for uni-directional translocation, and thus
ATP is not required in the latter type of flipping [138]. The polymerization reaction is chemically
similar to the reactions in the homopolymeric O-antigen, and N- and O-linked glycosylation
pathways, since all pathways rely on transfer of the glycan substrates from undecaprenol carrier
after translocation. However, the periplasmic polymerization of wzx-dependent undecaprenol
derivatives requires higher concentrations of periplasmic-oriented precursors, and thus an
equilibrative-based mechanism is efficient for facile transport of these substrates. In contrast, the
biosynthesis of homopolymeric O-antigen and N- and O-linked protein glycans does not involve
polymerization after translocation. This implies that the concentration of undecaprenylphosphate glycans may be too low for an equilibrative mechanism to be effective, and thus ATP
is needed to drive the translocation. Further work is required to understand the biological
necessity of the two distinct mechanistic classes of flippases.
22
While the identification of most bacterial flippases has been simplified due to gene
clustering in bacterial genomes, detailed biochemical characterization is still lacking in many
cases. For example, discovery of the enzyme responsible for Lipid II translocation (Fig. 5),
which is required for peptidoglycan biosynthesis, has been a significant challenge, despite the
large amount of research focused on this pathway during the last fifty years. Studies have
implicated both MviN and FtsW in Lipid II translocation [139, 148-151], but recent biochemical
evidence demonstrated that the presence of purified FtsW induced movement of Lipid II across
the bilayer in model membranes, suggesting that FtsW is the Lipid II flippase [139].
Protein-based mechanisms of translocation
Limited information is available on the mechanism of flippase-mediated translocation,
because, until recently, no biochemical methods for assaying translocation had been reported.
Developing a biochemical assay for monitoring flippase activity has been challenging, as it
requires a model membrane system and an assay specific for the polyprenol derivative. In
addition, polyprenol derivatives are hydrophobic molecules that can be difficult to quantify due
to weak UV absorbance, and flippases possess many transmembrane domains rendering them
relatively intractable to biochemical purification. However, in recent years, several important
studies exploiting lectin binding, as well as chemical-reporter and FRET-based methods for
quantifying the localization of polyprenyl-phosphate derivatives within membrane vesicles have
been reported [139, 140, 142, 147]. With the exception of the fluorescence-based method, which
requires a non-native glycan substrate, these methods are limited by the timescale of
visualization since flipping occurs faster than the time resolution of the assays, thereby
complicating mechanistic studies. In addition, there are no X-ray crystal structures of polyprenyl-
23
diphosphate glycan flippases, although the structure of a lipid flippase with homology to the
ATP-binding cassette flippases has been recently published [152].
Several studies have suggested mechanisms for flippase-mediated translocation. For
example, in a recent study that identified a protein fraction associated with Dol-P-Man
translocation activity, the authors found that citronellyl-phosphate, a short C10 dolichylphosphate analog, inhibited translocation [140]. This led to the suggestion that the flippase might
be also involved in translocation of dolichyl-phosphate as well as Dol-P-Man [140]. In this
model, the flippase acts as a pore through which the populations of Dol-P-Man and dolichylphosphate equilibrate, with Dol-P-Man moving to the ER lumen to act as a glycan donor and
dolichyl-phosphate returning to the cytoplasmic leaflet to begin a new cycle of glycan
biosynthesis. This model predicts that the cytoplasmic loops of the flippase enzyme might
preferentially recognize Dol-P-Man, whereas the periplasmic loops may prefer dolichylphosphate, which would help facilitate the desired flow of molecules, but this model is highly
speculative.
Another recent study utilized an unbiased truncation strategy to map the membrane
topology of the Wzx flippase involved in O-antigen biosynthesis in Pseudomonas aeruginosa
[141]. This study established that the flippase contained 12 transmembrane domains, similar to
flippases from eukaryotic pathways, and had several sizeable cytoplasmic loops, which may be
involved in recognition of the O-antigen glycan portion of the substrate. In addition, the
transmembrane helices included more charged residues than are typical for transmembrane
domains. This observation prompted the authors to propose that these mostly cationic residues
could line a channel formed by the transmembrane domains to facilitate movement of the O-
24
linked glycan across the membrane [141]. This study provides the basis for future mutational and
structural studies focused on understanding how flippases mediate translocation.
Potential role of polyprenol moieties in glycan translocation
In the studies presented above, mechanistic analysis of flippase reactions focused on the
protein catalysis, but it is also important to consider the specific role of the polyisoprenol
substrates. In particular, polyisoprenol recognition sequences (PIRS) are amino acid sequences
present in predicted transmembrane domains that have been shown to selectively bind
polyprenyl-phosphate-linked substrates in model membranes [107, 108]. PIRS were first
identified as dolichol recognition sequences (DRS) in the predicted transmembrane domains of
three essential glycosyltransferases in the yeast N-linked glycosylation pathway and were
postulated to interact with dolichol [153]. Subsequent studies identified PIRS in other essential
eukaryotic glycosyltransferases and E. coli enzymes involved in capsular polysaccharide
biosynthesis [154, 155]. The importance of these sequences is unclear as PIRS was found to be
essential for GlcNAc-1-phosphate transferase activity, but not for dolichyl-phosphate-mannose
synthase activity [156, 157].
Recent studies have utilized NMR and molecular modeling to study the binding of short
PIRS peptides to undecaprenol and C95-dolichol and the related phosphorylated derivatives
[107, 108]. These studies have proposed that polyprenols and polyprenyl-phosphates adopt a
coiled structure composed of three separate domains such that the head to tail length is shorter
than the width of the membrane (32-33 Å for C95-dolichol and 22 Å for undecaprenol) [108]
(Fig. 9), which is consistent with the overall architecture determined in a prior study on the
conformation of dolichol [109]. Furthermore, conformational analysis of how polyprenols may
interact with PIRS-based peptides suggested that multiple (4-5) peptides might be able to bind
25
each polyprenol molecule (Fig. 9). The authors propose that polyprenyl-phosphate may promote
recruitment of PIRS in nearby protein transmembrane domains and hence, the transient
formation of a hydrophilic channel appropriate for glycan translocation [107].
The flippase and PIRS hypotheses for glycan translocation are not mutually exclusive.
The PIR-sequence is not well defined, but a key element of the consensus sequence is the
presence of a proline [107, 108]. Prolines are rarely found in transmembrane domains, but are
frequently found in the transmembrane domains of transporter-related enzymes [158]. Indeed,
many of the flippase enzymes discussed above contain at least one transmembrane domain with a
proline residue. This could suggest that the proposed PIRS-polyisoprenol interaction may have a
role in the flippase mechanism, but further biochemical and biophysical studies are necessary to
elucidate the potential roles of these domains.
Why polyprenyl-phosphate? Models of macromolecular complexes
Here we consider a third hypothesis, which centers on the potential role of polyprenylphosphates in promoting the association of glycan biosynthetic enzymes. Both experimentally
established and hypothetical macromolecular enzyme complexes will be evaluated in light of the
essential polyprenyl-phosphate substrates. Several important multi-enzyme complexes that rely
on translocated polyprenyl-phosphate-linked substrates have been characterized, including the
eukaryotic oligosaccharyltransferase (OTase) and peptidoglycan biosynthetic machinery in
bacteria, although the role of polyprenols in the activities of these complexes remains unclear.
Multi-enzyme complexes with polyprenyl-phosphate-linked substrates
The eukaryotic OTase is a multimeric membrane-bound enzyme [27, 28, 159, 160], and
catalyzes transfer of the GlcNAc2Man9Glc3 tetradecasaccharide from dolichyl-diphosphate to
26
protein. With regards to the polyisoprenols, very little is known about how the dolichol moiety
interacts with the OTase complex, although the Wbp1 subunit has been suggested to bind a
dolichol derivative [161]. In vitro reconstitution of the OTase complex within a membranemimetic system would be one way to determine if dolichol derivatives are important for complex
formation and stability, however, this is extremely challenging due to the complexity of the
membrane-bound subunits in OTase.
In many ways, the eukaryotic OTase complex is unique among polyprenyl-phosphate
dependent pathways, because it is composed of multiple protein subunits acting in concert to
perform one reaction. Most other pathways dependent on polyprenols involve multiple
biosynthetic steps carried out by separate enzymes. For example, 15-20 different proteins have
been identified in the bacterial enzymatic machinery required for peptidoglycan biosynthesis [10,
51]. Recent studies have identified essential protein-protein interactions that suggest the
formation of both an elongase complex, responsible for lateral growth of rod-like bacteria, and a
distinct divisome complex, which mediates peptidoglycan synthesis at the site of cell division
[51]. Interestingly, both of these complexes are associated with a cytoskeletal protein (MreB or
FtsZ), which polymerizes at the cytoplasmic face of the cell surface and appears to recruit the
required protein components. In cell elongation, MreB forms a helical pattern on the cell surface,
whereas in cell division, FtsZ forms a ring at the center of rod-like cells to signal the site of
division. The elongase and divisome complexes require Lipid II to build peptidoglycan, but it is
not known if Lipid II itself is involved in organizing and recruiting the elongase and divisome
molecular machines. As discussed above, the relatively low pools of Lipid I/II found in Gramnegative bacteria suggests that cells have found a way to localize these substrates to sites of
27
synthesis, but little is known about how this may occur in Gram-negative or Gram-positive
bacteria.
Putative enzyme complexes reliant on polyprenyl-phosphate
Although many studies have elucidated models of the eukaryotic OT complex and
peptidoglycan biosynthesis, essentially nothing is known about enzyme complex formation in
other polyprenyl-phosphate dependent glycan assembly pathways. Identification of multienzyme biosynthetic complexes has been complicated since the protein-protein interactions may
be weaker than in the OTase complex and are not associated with a cytoskeletal protein as in
peptidoglycan biosynthesis. Glycan biosynthesis in eukaryotes and prokaryotes involves
sequential addition of individual glycan units to an undecaprenyl-phosphate carrier by distinct
enzymes (Fig. 3). Thus, macromolecular enzyme association is an attractive model for how
pathways may efficiently assembly polyprenyl-phosphate-linked glycans (Fig. 10). Furthermore,
polyprenyl-phosphate-linked substrates may act as more than passive glycan carriers by altering
membrane fluidity and specifically promoting both enzyme complex formation and substrate
flux through the pathway. The hypothesis of macromolecular complexes is not new, but previous
evidence is scant. As described above, examination of the interplay between teichoic acid and
peptidoglycan biosynthesis in Bacillus licheniformis led the authors to suggest that the
undecaprenyl-phosphate was sequestered by separate biosynthetic complexes [130]. Further
experimental evaluation of this hypothesis is challenging and may require in vitro reconstitution
of a polyprenol-based pathway in a membrane-mimetic system, and to our knowledge, no such
experiments have been performed to date.
As described above, the identification of specific interactions between PIRS peptides and
polyisoprenyl molecules was initially linked to polyprenyl-phosphate translocation [107, 108]. A
28
second interpretation, relevant here, is that the proposed ability of polyprenols to bind multiple
PIRS domains is involved in facilitating protein-protein interactions within a biosynthetic
pathway. This model suggests that polyprenols play an important role in recruitment of
biosynthetic enzymes into a macromolecular complex (Figs. 9 and 10), which could also have
important implications for how the polyprenyl-phosphate-linked glycan intermediates are
shuttled through the pathway.
Conclusion
Polyprenols and polyprenyl-phosphates are ubiquitous constituents of cellular membranes
and play roles as substrates in essential biosynthetic pathways in both eukaryotes and
prokaryotes. Much less is known about unmodified polyprenols, which act as precursors to
polyprenyl-phosphates in developing cells, but have no other established function in biological
systems and may be unnecessary in Gram-negative bacteria. Furthermore, polyprenols can
accumulate at relatively high levels within many eukaryotic organisms as well as Gram-positive
bacteria; biosynthesis of these molecules is a complex, energy-dependent process and it seems
unlikely polyprenol production would have been conserved in evolution if it did not serve an
important role. Further work is necessary to establish the physical effects that polyprenols may
exert on complex biological membranes and how polyprenol accumulation affects aging, disease,
and intracellular transport processes.
In contrast, the role of polyprenyl-phosphates as facilitators of glycan assembly in
essential bioprocesses such as N-linked protein glycosylation and peptidoglycan biosynthesis has
been extensively studied. Dolichyl-phosphates may serve even greater purposes by influencing
enzyme and pathway regulation, flippase-mediated glycan translocation across membranes, and
29
macromolecular enzyme complex formation. From these three hypotheses, it is interesting to
consider which better explains the evolutionary conservation of polyprenols and dolichols. The
regulatory role of dolichyl-phosphates is well established in eukaryotes, and it is certainly
important that dolichyl-phosphates have independent biosynthetic and recycling pathways from
other membrane components. However, it is not clear that the long, complex structure of
polyisoprenols is necessary for regulatory function. In contrast, the ubiquitous use of polyprenols
for translocation of glycans and their potential role in facilitating enzyme complex formation
offer much more tantalizing implications. Both of these processes involve localization and
movement within the lipid bilayer, processes that may be facilitated by the known biophysical
effects exerted by polyisoprenols and by the specific interactions that occur between
polyisoprenols and certain peptide sequences. Recent biochemical characterization of several
polyprenyl-phosphate dependent pathways [43, 50, 57, 58] coupled with exciting new membrane
mimetic technologies [162, 163] provide the tools for future biochemical and biophysical-based
elucidation of how polyprenyl-phosphates participate in glycan translocation and biosynthetic
complex assembly.
Acknowledgements
We gratefully acknowledge the valuable insights of Dr. Angelyn Larkin with regards to this
manuscript and thank Marcie Jaffee for her thoughtful comments. In addition, we would like to
acknowledge the National Institutes of Health (GM039334) for support of research related to the
content of this review in the Imperiali laboratories.
30
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Figure
Figure Captions:
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. (Top) Fully
unsaturated polyprenols contain all trans isoprene units or a mixture of trans and cis units as shown.
(Bottom) Dolichols are distinguished by a single saturated α-isoprene unit.
Fig. 2. Polyisoprene 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 step of dolichol biosynthesis involves
enzyme-catalyzed reduction of the α-isoprene subunit, which is thought to occur on the unmodified
polyprenol. Specific phosphatases responsible for the hydrolysis of dolichyl-diphosphate have not yet
been identified.
Fig. 3. Schematic of N-linked protein glycosylation pathways.
(Left) The eukaryotic pathway results in the assembly of a dolichyl-diphosphate (Dol-PP)
tetradecasaccharide 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
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 the arrow indicates 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-PPheptasaccharide and Dol-PP-tetradecasaccharide. (Lower right) The archaeal membrane contains Dol-PMan and dolichyl-phosphate-linked glycan precursors to N-linked glycans. The glycan produced in
Methanococcus voltae is shown, and 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 the arrow indicates 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 Grampositive bacteria such as 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 from E. coli strain O113, and homopolymeric O-antigens from Klebsiella
pneumoniae.
Fig. 6. Activated sugar donors. (Top) Dol-P-Glc is the membrane-bound sugar donor in the ER lumen of
eukaryotes. (Bottom) 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
containing a polyprenyl-phosphate glycan and polyprenyl-phosphate perpendicular to the membrane,
while polyprenol and esterified polyprenol parallel to the membrane surface.
Fig. 8. Cellular sources of dolichyl-phosphate. Dolichol is phosphorylated de novo by a kinase (K) to
generated dolichyl-phosphate or a salvage pathway occurs in which dolichyl-diphosphate is hydrolyzed
by a pyrophosphatase (P) and then translocated across the membrane by a flippase (F) to begin another
round of glycan biosynthesis. Dolichyl-diphosphate is the intermediate released after glycan transfer to
protein.
Fig. 9. Structures of dolichol, dolichyl-phosphate and peptide-bound dolichol. (A) From left to right,
structures of undecaprenyl-phosphate, C95 dolichyl-phosphate, and C95 dolichol were determined by
NMR and molecular modeling. The red group represents the phosphate. (B and C) 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. Images reproduced with permission from [108] and [107], respectively.
Fig. 10. 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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Table
Table 1. Structures of polyprenols in representative species.
a
Species
n
m
Ref
-saturated? Extended length
Bacteria
2
8
No
50 Å
[12]
Mycobacterium smegmatis
3b
3
No
34 Å
[16, 17]
1
8
No
46 Å
Halobacterium halobium (archaea)
2
9
Yes
60 Å
[13]
Saccharomyces cerevisiae
2 12-13
Yes
73-77 Å
[14]
Rattus norvegicus (rat)
2 13-16
Yes
76-89 Å
[15]
Homo sapiens
2 14-17
Yes
81-94 Å
[15]
Ficus elastica (plant)
3
6-8
No
47-56 Å
[22]
Aspergillus fumigatus (plant)
2 14-20
Yes
86-107 Å
[20]
Potentilla crinita (plant)
3 15-17
No
86-94 Å
[21]
3 25-27
129-137 Å
3 35-37
172-180 Å
a
Extended length refers to the predicted length of the polyprenol in an extended conformation.
b
The four isoprene units at the ω-terminal end are fully saturated in this short polyprenol from
Mycobaterium smegmatis. Two types of polyprenols are found in this bacterium.
Table 2. Intracellular quantification of dolichol and dolichyl-phosphate in mammalian sources.
Unless otherwise indicated, methods based on high performance liquid chromatography were
used to quantify the molecules (given in μg dolichol per g of cells).
Species
Dolichol
Dolichyl-phosphate
Ref
74 cpma
740 cpma
[69]
CHO (Chinese
hamster ovary) cells
690 cpma
270 cpma
[1]
a
a
270 cpm
59 cpm
[66]
Rat liver
43000 cpmb
890 cpmb
[66]
43.0 μg/g
8.75 μg/g
[15]
41 μg/g
9.38 μg/g
[65]
17.1 μg/g
14.7 μg/g
[68]
21.1 - 52.2 μg/gc
22.0 - 16.7 μg/gc
[67]
4.3 μg/g
16.0 μg/g
[68]
Rat testes
9.36 - 27.9 μg/gc
15.3 - 19.8 μg/gc
[67]
39.3 μg/g
18.7 μg/g
[68]
Rat spleen
58.9 - 157 μg/gc
11.6 - 24.8 μg/gc
[67]
11.3 μg/g
16.2 μg/g
[68]
Rat brain
9.36 - 27.9 μg/gc
15.3 - 19.8 μg/gc
[67]
7.8 μg/g
20.0 μg/g
[68]
Rat kidney
12.3 - 24.1 μg/gc
22.0 - 20.0 μg/gc
[67]
465 μg/g
10.8 μg/g
[15]
Human liver
486 μg/g
11.9 μg/g
[65]
1493 μg/g
169 μg/g
[15]
Human testis
a
Incorporation of radiolabeled mevalonate was used for quantification.
b
Incorporation of radiolabeled acetate was used for quantification.
c
In this study, the dolichol and dolichyl-phosphate amounts were determined at 4, 6, 8, 10 and
14 weeks; the values for 4 and 14 weeks are shown.
Table 3. The percentages of undecaprenol, undecaprenyl-phosphate (Und-P), and undecaprenyldiphosphate (Und-PP) in various bacterial species.
Species
Undecaprenol
Und-P
Und-PP
Reference
a
b
b
E. coli
nd
22% (75 nmol/g)
78% (260 nmol/g)
[89]
S. aureus
31% (70 nmol/g)
19% (43 nmol/g)b
50% (113 nmol/g)b
[89]
c
90%
<10%
n/a
[12]
S. faecalis
82%
18%
n/ac
[90]
a
No significant amounts of undecaprenol were detected.
b
The amount is given in nmoles polyprenol per gram cell pellet.
c
For the latter two studies, no attempt was made to quantify the amount of Und-PP.