Study of the N-glycoprotein biosynthesis through dolichol intermediates in the mitochondrial membranes

1. In the mitochondria, the biosynthesis of N-glycoprotein products, through the dolichol intermediates pathway, appears in the outer and in the inner membranes. 2. The biosynthesis of dolichol-pyrophosphoryl-N-acetyl-glucosamine, dolichol-pyrophosphoryl-di-N-acetylchitobiose, dolichol-phosphoryl-glucose and dolichol-phosphoryl-mannose is effective in both membranes. 3. The lipid-linked oligosaccharides biosynthesized in both membranes contain high mannose-type oligosaccharides ranging in size from Man9-GlcNac2 to Man4-GlcNac2. 4. The assembly of the dolichol-pyrophosphoryl-oligosaccharides on the trimannosidic core begins by the elongation of the alpha-1,3 mannose branch in the outer membrane and of the alpha-1,6 mannose branch in the inner membrane.     

Characterization of the submitochondrial compartments: study of the site of synthesis of dolichol and dolichol-linked sugars

The fractionation of mitochondrial membranes on discontinuous sucrose gradient leads to the obtaining of free outer membranes, free inner membranes and two distinct membrane contact site populations characterized as follows. Only outer membrane contact sites and inner membrane contact sites bind hexokinase. Outer membranes and outer membrane contact sites are cholesterol-rich fractions. The endogenous dolichol content is twice fold higher in outer membranes and outer membrane contact sites than in inner membranes and inner membrane contact sites, only the biosynthesis of dolichol in inner membrane contact sites is not stimulated by addition of exogenous [14C]-IPP and FPP. The glycosylation of endogenous dolichol from labeled nucleotide-sugars (UDP-GlcNAc, GDP-Man and UDP-Glc) leads to the synthesis of dolichol-pyrophosphoryl-sugars and dolichol-monophosphoryl-sugars with the rate of synthesis proportional to the dolichol content of each submitochondrial fraction.     

Synthesis of N- and O-linked glycopeptides in oviduct membrane preparations

A hen oviduct membrane preparation that catalyzes both the N- and O-glycosylation of exogenous acceptor peptides was used to examine the possible involvement of lipid intermediates in enzymatic O-glycosylation. The results indicate that, under a variety of experimental conditions in which the dolichol-linked saccharides involved in N-glycosylation are readily observed, no lipid-linked intermediates for O-glycosylation could be detected. Whereas N-glycosylation is abolished by tunicamycin treatment and stimulated by dolichol phosphate addition, O-glycosylation is unaffected by such treatments. Further, the results of subcellular fractionation of oviduct membranes suggest that N-acetylgalactosaminyl:polypeptide transferase is localized primarily in membranes derived from the smooth endoplasmic reticulum and Golgi apparatus. This is in contrast to the subcellular site of N-glycosylation, which has previously been shown to be primarily the rough endoplasmic reticulum. These findings are discussed in relation to the function of dolichol phosphate in protein glycosylation.     

Distribution of glycosyltransferase activities in different compartments of mitochondria

The liver mitochondria were submitted to a first swelling which allowed to get outer membranes. The mitoplasts obtained in these conditions were subject to a second swelling. The separation of submitochondrial membranes on a discontinuous sucrose gradient revealed three membrane fractions, an outer membrane rich fraction, an inner membrane rich fraction and a fraction enriched with contact sites between the two membranes. The various glycosyltransferase systems involved in the biosynthesis of N-glycoproteins were located in these fractions.     

Identification of phosphorylated oligosaccharides in cells of patients with a congenital disorders of glycosylation (CDG-I)

Protein N-glycosylation is initiated by the dolichol cycle in which the oligosaccharide precursor Glc₃Man₉GlcNAc₂-PP-dolichol is assembled in the endoplasmic reticulum (ER). One critical step in the dolichol cycle concerns the availability of Dol-P at the cytosolic face of the ER membrane. In RFT1 cells, the lipid-linked oligosaccharide (LLO) intermediate Man₅GlcNAc₂-PP-Dol accumulates at the cytosolic face of the ER membrane. Since Dol-P is a rate-limiting intermediate during protein N-glycosylation, continuous accumulation of Man₅GlcNAc₂-PP-Dol would block the dolichol cycle. Hence, we investigated the molecular mechanisms by which accumulating Man₅GlcNAc₂-PP-Dol could be catabolized in RFT1 cells. On the basis of metabolic labeling experiments and in comparison to human control cells, we identified phosphorylated oligosaccharides (POS), not found in human control cells and present evidence that they originate from the accumulating LLO intermediates. In addition, POS were also detected in other CDG patients’ cells accumulating specific LLO intermediates at different cellular locations. Moreover, the enzymatic activity that hydrolyses oligosaccharide-PP-Dol into POS was identified in human microsomal membranes and required Mn²⁺ for optimal activity. In CDG patients’ cells, we thus identified and characterized POS that could result from the catabolism of accumulating LLO intermediates.     

Mitochondrial hexokinase from small-intestinal mucosa and brain

1. The submitochondrial localization of hexokinase activity in preparations of mitochondria from the small intestine of the guinea pig was studied by conventional methods. 2. Hexokinase activity in this tissue was predominantly associated with the outer mitochondrial membrane. 3. The inactivation of mitochondrial enzymes by trypsin in iso-osmotic and hypo-osmotic conditions was also used to determine the submitochondrial localization of hexokinase activity. 4. Hexokinase activity was found to be on the outside of the outer mitochondrial membrane. 5. It was shown that both type I and type II hexokinase activities are bound to the outside of the outer mitochondrial membrane. The types are present in the same ratio as that in which they occur in the cytosol of the cell. 6. Mitochondrial hexokinase from the small intestine did not show the latency phenomenon demonstrated by mitochondrial hexokinase from brain when subjected to a variety of treatments. However, hexokinase activity was solubilized from preparations of mitochondria from the small intestine by the same treatments as for mitochondrial hexokinase from brain. 7. The submitochondrial distribution of hexokinase activity in mitochondrial preparations from rat brain was determined by the trypsin inactivation method. 8. Hexokinase activity in preparations of mitochondria from rat brain was found on the outside of the outer membrane, between the mitochondrial membranes, and within the inner mitochondrial membrane. 9. Hexokinase from rat brain showed latency properties irrespective of its submitochondrial location.     

The tissue and subcellular distribution of [3H]dolichol in the rat

Following the intravenous injection of nanomolar amounts of [³H]dolichol into rats, the radioactivity rapidly appeared in the high-density lipoprotein fraction of the plasma and circulated with a half-life of about 9 h. A fraction of the injected activity was excreted in the feces, presumably through the bile, but evidence was obtained that little oxidative breakdown of dolichol occurred. All tissues assayed acquired radioactivity, but the liver attained the highest specific activity and the largest percentage of the total radioactive dolichol. Subcellular fractionation of the liver revealed that mitochondrial preparations contained the bulk of the labeled dolichol at all times tested up to 40 h after injection. Disruption of the mitochondrial structure by two different techniques permitted the isolation of inner and outer membrane fractions and it was found that the [³H]dolichol was concentrated in the outer membrane fraction. The significance of these findings is discussed.     

Mitochondrial protein import: differential recognition of various transport intermediates by antibodies

The precursors of the mitochondrial proteins ADP/ATP carrier (AAC) and F1-ATPase subunit beta (F1 beta) were accumulated at the stages of binding to receptor sites on the mitochondrial outer membrane, or in contact sites between outer and inner membranes. Specific antibodies raised against the mature proteins were added to the isolated mitochondria and efficiently bound to these translocation intermediates. Further movement of the precursors to consecutive steps along their import pathway was thereby inhibited. Controls showed that precursor proteins which were inserted into or translocated across the outer membrane were not recognized by the antibodies unless the mitochondrial membranes were disrupted. We conclude that the trapped translocation intermediates have antigenic sites exposed to the outside of the outer membrane.     

Subcellular and submitochondrial localization of phospholipid-synthesizing enzymes in Saccharomyces cerevisiae.

Using highly enriched membrane preparations from lactate-grown Saccharomyces cerevisiae cells, the subcellular and submitochondrial location of eight enzymes involved in the biosynthesis of phospholipids was determined. Phosphatidylserine decarboxylase and phosphatidylglycerolphosphate synthase were localized exclusively in the inner mitochondrial membrane, while phosphatidylethanolamine methyltransferase activity was confined to microsomal fractions. The other five enzymes tested in this study were common both to the outer mitochondrial membrane and to microsomes. The transmembrane orientation of the mitochondrial enzymes was investigated by protease digestion of intact mitochondria and of outside-out sealed vesicles of the outer mitochondrial membrane. Glycerolphosphate acyltransferase, phosphatidylinositol synthase, and phosphatidylserine synthase were exposed at the cytosolic surface of the outer mitochondrial membrane. Cholinephosphotransferase was apparently located at the inner aspect or within the outer mitochondrial membrane. Phosphatidate cytidylyltransferase was localized in the endoplasmic reticulum, on the cytoplasmic side of the outer mitochondrial membrane, and in the inner mitochondrial membrane. Inner membrane activity of this enzyme constituted 80% of total mitochondrial activity; inactivation by trypsin digestion was observed only after preincubation of membranes with detergent (0.1% Triton X-100). Total activity of those enzymes that are common to mitochondria and the endoplasmic reticulum was about equally distributed between the two organelles. Data concerning susceptibility to various inhibitors, heat sensitivity, and the pH optima indicate that there is a close similarity of the mitochondrial and microsomal enzymes that catalyze the same reaction.     

Overexpression of the Saccharomyces cerevisiae RER2 gene in Trichoderma reesei affects dolichol dependent enzymes and protein glycosylation

Protein secretion in Trichoderma reesei could be stimulated by overexpression of the yeast Saccharomyces cerevisiae DPM1 gene encoding dolichyl phosphate mannose synthase (DPMS) a key enzyme in the O-glycosylation pathway. The secreted proteins were glycosylated to the wild type level. On the other hand, the elevated concentration of GDP-mannose, a direct substrate for DPMS, resulting from overexpression in T. reesei of the mpg1 gene coding for guanyltransferase, did not affect secretion of proteins but did affect the degree of their O- and N-glycosylation. In this paper, we examined the effects of dolichol, an indispensable carrier of sugar residues in protein glycosylation, on the synthesis of glycosylated proteins. An increase in dolichol synthesis was obtained by overexpression of the yeast gene encoding cis-prenyltransferase, the first enzyme of the mevalonate pathway committed to dolichol biosynthesis. We observed that, an increased concentration of dolichol resulted in an increased expression of the dpm1 gene and DPMS activity and in overglycosylation of secreted proteins.     

Characterization of translocation contact sites involved in the import of mitochondrial proteins

Import of proteins into the mitochondrial matrix requires translocation across two membranes. Translocational intermediates of mitochondrial proteins, which span the outer and inner membrane simultaneously and thus suggest that translocation occurs in one step, have recently been described (Schleyer, M., and W. Neupert, 1985, Cell, 43:339-350). In this study we present evidence that distinct membrane areas are involved in the translocation process. Mitochondria that had lost most of their outer membrane by digitonin treatment (mitoplasts) still had the ability to import proteins. Import depended on proteinaceous structures of the residual outer membrane and on a factor that is located between the outer and inner membranes and that could be extracted with detergent plus salt. Translocational intermediates, which had been preformed before fractionation, remained with the mitoplasts under conditions where most of the outer membrane was subsequently removed. Submitochondrial vesicles were isolated in which translocational intermediates were enriched. Immunocytochemical studies also suggested that the translocational intermediates are located in areas where outer and inner membranes are in close proximity. We conclude that the membrane-potential-dependent import of precursor proteins involves translocation contact sites where the two membranes are closely apposed and are linked in a stable manner.     

Dolichol kinase activity: a key factor in the control of N-glycosylation in inner mitochondrial membranes

Inner mitochondrial membranes from liver contain a dolichol kinase which required CTP as a phosphoryl donor. Kinase activity was linear with protein concentration and unlike other reported kinases, activated almost equally well by Mg2+, Mn2+ or Ca2+. Thin-layer chromatography showed that the reaction product co-migrated with authentic dolichyl monophosphate. The phosphorylation of dolichol did not occur in presence of ATP, GTP or UTP but required exogenous dolichol for maximal activity. Newly synthesized [3H]dolichyl monophosphate has been shown to be glycosylated in the presence of GDP[14C]mannose or UDP[14C]glucose. The double labeled lipids formed by the sugar nucleotide-dependent reactions were identified respectively as [14C]mannosylphosphoryl[3H]dolichol and [14C]glucosylphosphoryl [3H]dolichol. These results are discussed in terms of regulation of N-glycosylation processes in inner mitochondrial membranes from liver.     

Biosynthesis of glycoconjugates in mitochondrial outer membranes

The results reported in this paper show two distinct ways for the incorporation ofN-acetylglucosamine into mitochondrial outer membranes. The first one is the glycosylation of dolichol acceptors, which is indicated by the inhibition of the synthesis of these products by the inhibitors of the dolichol intermediates (tunicamycin and GDP). The second one is the incorporation ofN-acetylglucosamine into protein acceptors directly from UDP-N-acetylglucosamine. This second way of glycosylation is only localized in mitochondria outer membranes.The existence of a direct route forN-glycoprotein biosynthesis has been based on the following evidence. First, the synthesis of theN-acetylglucosaminylated protein acceptors was not inhibited by tunicamycin or GDP. Second, the addition of exogenous dolichol-phosphate did not change the rate of biosynthesis of glycosylated protein material. Third, the sequential incorporation ofN-acetylglucosamine and mannose from their nucleotide derivatives in the presence of GDP and tunicamycin led to the synthesis of glycosylated protein material which entirely bound to Concanavalin A-Sepharose. The oligosaccharide moiety of the glycosylated protein material resulting from the direct transfer of sugars from their nucleotide derivatives to the protein acceptor is of theN-glycan type. On sodium dodecylsulphate polyacrylamide gel electrophoresis, this glycosylated material migrated as a marker protein with a molecular weight between 45 000 and 63 000. HPLC chromatofocusing analysis revealed that the fraction studied was anionic. The oligosaccharide moiety of the glycoprotein material can only be elongated by the incorporation ofN-acetylglucosamine and galactose from their nucleotide derivatives.     

Esterification of dolichol in rat liver

In the various subcellular fractions of rat liver 45-75% of the total dolichol was esterified with a fatty acid. The esterification reaction was localized exclusively in the microsomes, and the transferase activity is 3-fold higher in the cation-insensitive smooth microsomes than in other microsomal subfractions. Although fatty acyl-CoAs tested served as substrates, palmitoyl-CoA was the most rapidly utilized. None of the phosphatidylcholine or phosphatidylethanolamine species tested could be utilized to esterify dolichol with a fatty acid, indicating the absence of transacylation. alpha-Saturated dolichols were esterified at a higher rate than their alpha-unsaturated counterparts. Albumin and low concentrations of Triton X-100 activated the esterification reaction, which was not dependent on mono- or divalent cations, ATP, or CoA. The sensitivity of the transferase activity to trypsin indicates localization of the enzyme(s) involved on the outer surface of microsomes (i.e. the cytoplasmic surface of the endoplasmic reticulum), as is also the case for enzymes of dolichol biosynthesis. Transferase activity was detected in all tissues examined but at a much lower level than in liver and testis. The patterns of fatty acids in dolichol esters of different organelles exhibited some specificity. Labeling in vivo indicated that esterification of dolichol may play a role in targeting this lipid from the endoplasmic reticulum to lysosomes.     

The topography of glycerophosphate acyltransferase in the transverse plane of the mitochondrial outer membrane

In low ionic media, mitochondrial glycerophosphate acyltransferase was inhibited virtually completely within 15 min by the nonspecific proteases, proteinase K and subtilisin. In high ionic media, the mitochondrial enzyme was either not inhibited or was marginally inhibited by these proteases. Chymotrypsin and trypsin, regardless of the ionic strength of the medium, did not inhibit the acyltransferase. Substantial inhibition by proteinase K and subtilisin was observed in the high ionic media when the incubation was continued for 30 or 45 min. Adenylate kinase, an intermembrane enzyme, was not inhibited under any of the above conditions. These results demonstrate a cytosolic exposure of the mitochondrial acyltransferase. In a low ionic environment, when the outer membrane integrity was damaged either by gradually decreasing the tonicity of the medium or by stepwise addition of Triton X-100, either chymotrypsin or trypsin caused virtually parallel inhibition of glycerophosphate acyltransferase and adenylate kinase. A more direct approach in establishing the existence of protease-susceptible sites on the inner side of the outer membrane was taken by observing the inhibition of mitochondrial glycerophosphate acyltransferase and adenylate kinase in trypsinloaded right-side-out outer membrane vesicles incubated in the presence of externally located soybean trypsin inhibitor. The above results, taken together, suggest that mitochondrial glycerophosphate acyltransferase spans the transverse plane of the outer membrane.     

Recycling of dolichyl monophosphate to the cytoplasmic leaflet of the endoplasmic reticulum after the cleavage of dolichyl pyrophosphate on the lumenal monolayer

During protein N-glycosylation, dolichyl pyrophosphate (Dol-P-P) is discharged in the lumenal monolayer of the endoplasmic reticulum (ER). Dol-P-P is then cleaved to Dol-P by Dol-P-P phosphatase (DPPase). Studies with the yeast mutant cwh8Delta, lacking DPPase activity, indicate that recycling of Dol-P produced by DPPase contributes significantly to the pool of Dol-P utilized for lipid intermediate biosynthesis on the cytoplasmic leaflet. Whether Dol-P formed in the lumen diffuses directly back to the cytoplasmic leaflet or is first dephosphorylated to dolichol has not been determined. Incubation of sealed ER vesicles from calf brain with acetyl-Asn-Tyr-Thr-NH(2), an N-glycosylatable peptide, to generate Dol-P-P in the lumenal monolayer produced corresponding increases in the rates of Man-P-Dol, Glc-P-Dol, and GlcNAc-P-P-Dol synthesis in the absence of CTP. No changes in dolichol kinase activity were observed. When streptolysin-O permeabilized CHO cells were incubated with an acceptor peptide, N-glycopeptide synthesis, requiring multiple cycles of the dolichol pathway, occurred in the absence of CTP. The results obtained with sealed microsomes and CHO cells indicate that Dol-P, formed from Dol-P-P, returns to the cytoplasmic leaflet where it can be reutilized for lipid intermediate biosynthesis, and dolichol kinase is not required for recycling. It is possible that the flip-flopping of the carrier lipid is mediated by a flippase, which would provide a mechanism for the recycling of Dol-P derived from Man-P-Dol-mediated reactions in N-, O-, and C-mannosylation of proteins, GPI anchor assembly, and the three Glc-P-Dol-mediated reactions in Glc(3)Man(9)GlcNAc(2)-P-P-Dol (DLO) biosynthesis.     

Cytochromes c1 and b2 are sorted to the intermembrane space of yeast mitochondria by a stop-transfer mechanism.

The pathway by which cytochromes c1 and b2 reach the mitochondrial intermembrane space has been controversial. According to the "conservative sorting" hypothesis, these proteins are first imported across both outer and inner membranes into the matrix, and then are retranslocated across the inner membrane. Our data argue against this model: import intermediates of cytochromes c1 and b2 were found only outside the inner membrane; maturation of these proteins was independent of the matrix-localized hsp60 chaperone; and dihydrofolate reductase linked to the presequence of either cytochrome was imported to the intermembrane space in the absence of ATP. We conclude that cytochromes c1 and b2 are sorted by a mechanism in which translocation through the inner membrane is arrested by a "stop-transfer" signal in the presequence. The arrested intermediates may be associated with a proteinaceous channel in the inner membrane.     

Hypoglycosylation due to dolichol metabolism defects

Dolichol phosphate is a lipid carrier embedded in the endoplasmic reticulum (ER) membrane essential for the synthesis of N-glycans, GPI-anchors and protein C- and O-mannosylation. The availability of dolichol phosphate on the cytosolic site of the ER is rate-limiting for N-glycosylation. The abundance of dolichol phosphate is influenced by its de novo synthesis and the recycling of dolichol phosphate from the luminal leaflet to the cytosolic leaflet of the ER. Enzymatic defects affecting the de novo synthesis and the recycling of dolichol phosphate result in glycosylation defects in yeast or cell culture models, and are expected to cause glycosylation disorders in humans termed congenital disorders of glycosylation (CDG). Currently only one disorder affecting the dolichol phosphate metabolism has been described. In CDG-Im, the final step of the de novo synthesis of dolichol phosphate catalyzed by the enzyme dolichol kinase is affected. The defect causes a severe phenotype with death in early infancy. The present review summarizes the biosynthesis of dolichol-phosphate and the recycling pathway with respect to possible defects of the dolichol phosphate metabolism causing glycosylation defects in humans.     

StAR search--what we know about how the steroidogenic acute regulatory protein mediates mitochondrial cholesterol import

Cholesterol is the starting point for biosynthesis of steroids, oxysterols and bile acids, and is also an essential component of cellular membranes. The mechanisms directing the intracellular trafficking of this insoluble molecule have received attention through the discovery of the steroidogenic acute regulatory protein (StAR) and related proteins containing StAR-related lipid transfer domains. Much of our understanding of the physiology of StAR derives from studies of congenital lipoid adrenal hyperplasia, which is caused by StAR mutations. Multiple lines of evidence show that StAR moves cholesterol from the outer to inner mitochondrial membrane, but acts exclusively on the outer membrane. The precise mechanism by which StAR's action on the outer mitochondrial membrane stimulates the flow of cholesterol to the inner membrane remains unclear. When StAR interacts with protonated phospholipid head groups on the outer mitochondrial membrane, it undergoes a conformational change (molten globule transition) that opens and closes StAR's cholesterol-binding pocket; this conformational change is required for cholesterol binding, which is required for StAR activity. The action of StAR probably requires interaction with the peripheral benzodiazepine receptor.     

Intramembranous arrangement of the glycosylating systems in rough and smooth microsomes from rat liver

The distribution of mannosyl-, glucosaminyl- and glucosyltransferases in rough and smooth microsomes isolated from rat liver homogenate has been investigated. Amphomycin and tunicamycin were used as inhibitors of dolichol-mediated glycosylation, and diazobenzene sulfonate and proteolytic enzymes were used as nonpenetrating surface probes. Under in vitro conditions only 20-30% of the proteins glycosylated are of the secretory type. Nonpenetrating surface probes, which interact with components on the outer surface of rough microsomal vesicles, decrease glycosylation of both secretory and membrane proteins to a great extent. Inhibitor sensitive glycosylation is present in both the outer and inner compartments of the microsomal membranes. In contrast, the surface probes and the inhibitors of dolichol-mediated glycosylation do not significantly affect protein glycosylation in smooth microsomes. When dolichol phosphate sugars were used as substrates, instead of nucleotide sugars, the probes used inhibited protein glycosylation in both subfractions. Glycosylation of externally added Lipidex-bound dolichol monophosphate and of ovalbumin were in agreement with the above results. It appears that both rough and smooth microsomes may possess several types of glycosylating pathways. The most prominent of these in rough microsomes under the conditions used is the dolichol mono- and pyrophosphate-mediated glycosylation of endogenous proteins, where the enzymes involved in the initial steps are distributed at the outer surfaces of the microsomal vesicles. The dominating pathway in smooth microsomes appears to function in completion of the oligosaccharide chain of the protein and this process does not involve lipid intermediates and cannot be influenced by nonpenetrating surface probes.