Protein O-mannosyltransferases associate with the translocon to modify translocating polypeptide chains.

O-Mannosylation and N-glycosylation are essential protein modifications that are initiated in the endoplasmic reticulum (ER). Protein translocation across the ER membrane and N-glycosylation are highly coordinated processes that take place at the translocon-oligosaccharyltransferase (OST) complex. In analogy, it was assumed that protein O-mannosyltransferases (PMTs) also act at the translocon, however, in recent years it turned out that prolonged ER residence allows O-mannosylation of un-/misfolded proteins or slow folding intermediates by Pmt1-Pmt2 complexes. Here, we reinvestigate protein O-mannosylation in the context of protein translocation. We demonstrate the association of Pmt1-Pmt2 with the OST, the trimeric Sec61, and the tetrameric Sec63 complex in vivo by co-immunoprecipitation. The coordinated interplay between PMTs and OST in vivo is further shown by a comprehensive mass spectrometry-based analysis of N-glycosylation site occupancy in pmtΔ mutants. In addition, we established a microsomal translation/translocation/O-mannosylation system. Using the serine/threonine-rich cell wall protein Ccw5 as a model, we show that PMTs efficiently mannosylate proteins during their translocation into microsomes. This in vitro system will help to unravel mechanistic differences between co- and post-translocational O-mannosylation.

Photoaffinity labeling of protein O-mannosyltransferases of the PMT1/PMT2 subfamily.

Protein O-mannosylation is initiated at the endoplasmic reticulum (ER) by dolichyl phosphate-mannose: protein O-mannosyltransferases (PMTs). PMTs are members of the glycosyltransferase (GT) C superfamily. They are large polytopic integral membrane proteins located in the ER membrane. PMTs utilize dolichyl phosphate--activated mannose as sugar donor. Glycosyltransfer of mannose to serine and threonine residues of nascent polypeptides leads to an inversion of the stereochemistry of the glycosidic bond. Here, we describe photoaffinity labeling of yeast Pmt1p using a photo-reactive probe that is based on the artificial mannosyl acceptor peptide YATAV. Due to the high homology of PMTs, this method can also be applied to study PMT1 and PMT2 subfamily members from fungi other than baker's yeast.

Cell growth-dependent coordination of lipid signaling and glycosylation is mediated by interactions between Sac1p and Dpm1p.

The integral membrane lipid phosphatase Sac1p regulates local pools of phosphatidylinositol-4-phosphate (PtdIns(4)P) at endoplasmic reticulum (ER) and Golgi membranes. PtdIns(4)P is important for Golgi trafficking, yet the significance of PtdIns(4)P for ER function is unknown. It also remains unknown how localization of Sac1p to distinct organellar membranes is mediated. Here, we show that a COOH-terminal region in yeast Sac1p is crucial for ER targeting by directly interacting with dolicholphosphate mannose synthase Dpm1p. The interaction with Dpm1p persists during exponential cell division but is rapidly abolished when cell growth slows because of nutrient limitation, causing translocation of Sac1p to Golgi membranes. Cell growth-dependent shuttling of Sac1p between the ER and the Golgi is important for reciprocal control of PtdIns(4)P levels at these organelles. The fraction of Sac1p resident at the ER is also required for efficient dolichol oligosaccharide biosynthesis. Thus, the lipid phosphatase Sac1p may be a key regulator, coordinating the secretory capacity of ER and Golgi membranes in response to growth conditions.

An efficient assay for dolichyl phosphate-mannose: protein O-mannosyltransferase.

A novel method for quantifying the reaction product from dolichyl phosphoryl mannose:polypeptide mannosyltransferase (protein mannosyl transferase; PMT), was developed. The assay quantifies the amount of radioactivity incorporated into the acceptor peptide YNPTSV from dolichyl phosphoryl [3H]mannose (Dol-P-Man). A novel delivery system, large unilamellar vesicles (LUV), composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), is used to keep the poorly soluble donor substrate, Dol-P-Man, in solution. The use of LUV allows generation of truly reproducible data and, as an additional benefit, also results in a more than 10 times increase in transfer efficiency. In contrast to the solvent extraction procedures commonly used in previously described PMT assays, the assay reaction product is separated from the radioactive donor substrate on C(18) cartridges. The use of C(18) cartridges allows generation of reproducible data with a low, consistent background and also produces a significant reduction in the time and labor needed for the product workup. In a reaction mixture consisting of 100 microg POPC LUV, 9 x 10(5)cpm (approximately 15 pmol) Dol-P-Man, 100 nmol YNPTSV, and aproximately 4 microg of crude yeast microsomal extract, time-dependent formation of glycosylated product obeys Michaelis-Menten-type kinetics throughout the course of the reaction-until exhaustion of the donor substrate. The linear initial rates of the reaction allowed calculation of an apparent K(m) of 1mM, for the acceptor peptide YNPTSV. Variations in detergent concentration in the assay influence transfer efficiency, possibly through interference with the LUV-based donor substrate delivery system. Hence detergent concentrations should be kept constant.

Proteins in the early golgi compartment of Saccharomyces cerevisiae immunoisolated by Sed5p.

The yeast tSNARE Sed5p is considered to mainly reside in the early Golgi compartment at the steady state of its intracellular cycling. To better understand this compartment, we immunoisolated a membrane subfraction having Sed5p on the surface (the Sed5 vesicles). Immunoblot studies showed that considerable portions (20-30%) of the Golgi mannosyltransferases (Mnt1p, Van1p, and Mnn9p) were simultaneously recovered while the late Golgi (Kex2p) or endoplasmic reticulum (Sec71p) proteins were almost excluded. The N-terminal sequences of the polypeptides detectable by Coomassie blue staining indicated that the prominent components of the Sed5 vesicles include Anp1p, Emp24p, Erv25p, Erp1p, Ypt52p, and a putative membrane protein of unknown function (Yml067c).

Golgi localization and functionally important domains in the NH2 and COOH terminus of the yeast CLC putative chloride channel Gef1p.

GEF1 encodes the single CLC putative chloride channel in yeast. Its disruption leads to a defect in iron metabolism (Greene, J. R., Brown, N. H., DiDomenico, B. J., Kaplan, J., and Eide, D. (1993) Mol. Gen. Genet. 241, 542-553). Since disruption of GEF2, a subunit of the vacuolar H+-ATPase, leads to a similar phenotype, it was previously suggested that the chloride conductance provided by Gef1p is necessary for vacuolar acidification. We now show that gef1 cells indeed grow less well at less acidic pH. However, no defect in vacuolar acidification is apparent from quinacrine staining, and Gef1p co-localizes with Mnt1p in the medial Golgi. Thus, Gef1p may be important in determining Golgi pH. Systematic alanine scanning of the amino and the carboxyl terminus revealed several regions essential for Gef1p localization and function. One sequence (FVTID) in the amino terminus conforms to a class of sorting signals containing aromatic amino acids. This was further supported by point mutations. Alanine scanning of the carboxyl terminus identified a stretch of roughly 25 amino acids which coincides with the second CBS domain, a conserved protein motif recently identified. Mutations in the first CBS domain also destroyed proper function and localization. The second CBS domain can be transplanted to the amino terminus without loss of function, but could not be replaced by the corresponding domain of the homologous mammalian channel ClC-2.

Mannolipid donor specificity of glycosylphosphatidylinositol mannosyltransferase-I (GPIMT-I) determined with an assay system utilizing mutant CHO-K1 cells.

The microsomal enzyme glycosylphosphatidylinositol mannosyltransferase I (GPIMT-I) catalyses the transfer of a mannosyl residue from beta-mannosylphosphoryldolichol (beta-Man-P-Dol) to glucosamine-alpha(1,6)(acyl)phosphatidylinositol (GlcN-aPI) to form Man alpha(1,4)GlcN-aPI (ManGlcN-aPI), an intermediate in glycosylphosphatidylinositol (GPI) synthesis. While the transfer of [3H]mannosyl units to endogenous GlcN-aPI was not seen when membrane fractions from normal Chinese hamster ovary (CHO) K1 cells were incubated with exogenous [3H]Man-P-Dol, GPIMT-I activity could be characterized with an in vitro enzyme assay system employing membrane fractions from Lec15 or Lec35 cells. These CHO cell mutants apparently contain elevated levels of endogenous GlcN-aPI due to the inability to synthesize (Lec15) or utilize (Lec35) beta-Man-P-Dol in vivo. The presence of a saturated alpha-isoprene unit in the dolichyl moiety is required for optimal GPIMT-I activity since beta-mannosylphosphorylpolyprenol (beta-Man-P-Poly), which contains a fully unsaturated polyisoprenyl chain, was only 50% as effective as beta-[3H]Man-P-Dol as a mannosyl donor. When beta-[3H]-Man-P-Dol and alpha-[3H]Man-P-Dol were compared as substrates, GPIMT-I exhibited a strict stereospecificity for the mannolipid containing the beta-mannosyl-phosphoryl linkage. beta-[3H]Man-P-dolichols containing 11 or 19 isoprenyl units were equally effective substrates for GPIMT-I. Membrane fractions from Lec 9, a CHO mutant that apparently lacks polyprenol reductase activity and synthesizes very little beta-Man-P-Dol, but accumulates beta-Man-P-Poly, synthesized no detectable Man-GlcN-aPI when incubated with beta-[3H]Man-P-Dol in vitro. This indirect assay suggests that GlcN-aPI does not accumulate in Lec 9 cells, possibly because it is mannosylated via beta-Man-P-Poly, or perhaps the small amount of Man-P-Dol formed by the mutant in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondrial dolichyl-phosphate mannose synthase. Purification and immunogold localization by electron microscopy.

Mitochondrial dolichyl-phosphate mannose synthase has been purified to homogeneity using an original procedure, reconstitution into specific phospholipid vesicles and sedimentation on a sucrose gradient as final step. The enzyme has an apparent molecular mass of 30 kDa on an SDS/polyacrylamide gel. Increased enzyme activity could be correlated with this polypeptide band. A specific antibody was raised in rabbits against this transferase. Specific IgG obtained from the immune serum removed enzymatic activity from a detergent extract of mitochondrial outer membrane and reacted specifically with the 30-kDa band on immunoblots. Furthermore, an immunocytochemical experiment proved the localization of dolichyl-phosphate mannose synthase on the cytosolic face of the outer membrane of mitochondria.

Yeast KEX2 protease and mannosyltransferase I are localized to distinct compartments of the secretory pathway.

The KEX2 protease (product of the KEX2 gene) functions late in the secretory pathway of Saccharomyces cerevisiae by cleaving the polypeptide chains of prepro-killer toxin and prepro-alpha-factor at paired basic amino acid residues. The intracellular vesicles containing KEX2 protease sedimented in density gradients to a position distinct from those containing mannosyltransferase I (product of the MNN1 gene), a marker enzyme for the Golgi complex. The recovery of intact compartments containing these enzymes approached 80% after sedimentation. We propose that the KEX2 protease and mannosyltransferase I reside within distinct compartments.

Mannosylation of endogenous and exogenous phosphatidic acid by liver microsomal membranes. Formation of phosphatidylmannose.

Hamster liver post-nuclear membranes catalyze the transfer of mannose from GDP-mannose to endogenous dolichyl phosphate and to a second major endogenous acidic lipid. This mannolipid was believed to be synthesized from endogenous retinyl phosphate and was tentatively identified as retinyl phosphate mannose (Ret-P-Man) (De Luca, L. M., Brugh, M. R. Silverman-Jones, C. S. and Shidoji, Y. (1982) Biochem. J. 208, 159-170). To characterize this endogenous mannolipid in more detail, we isolated and purified the mannolipid from incubations containing hamster liver membranes and GDP-[14C]mannose and compared its properties to those of authentic Ret-P-Man. We found that the endogenous mannolipid was separable from authentic Ret-P-Man on a Mono Q anion exchange column, did not exhibit the absorbance spectrum characteristic of a retinol moiety, and was stable to mild acid under conditions which cleave authentic Ret-P-Man. The endogenous mannolipid was sensitive to mild base hydrolysis and mannose was released from the mannolipid by snake venom phosphodiesterase digestion. These properties were consistent with the endogenous acceptor being phosphatidic acid. Addition of exogenous phosphatidic acid, but not phospholipids with a head group blocking the phosphate moiety, to incubations containing hamster liver membranes and GDP-[14C]mannose resulted in the synthesis of a mannolipid with chromatographic and physical properties identical to the endogenous mannolipid. A double-labeled mannolipid was synthesized in incubations containing hamster liver membranes, GDP-[14C]mannose, and [3H]phosphatidic acid. Mannosyl transfer to exogenous phosphatidic acid was saturable with increasing concentrations of phosphatidic acid and GDP-mannose and specific for glycosyl transfer from GDP-mannose. Class E Thy-1-negative mutant mouse lymphoma cell membranes, which are defective in dolichyl phosphate mannose synthesis, also fail to transfer mannose from GDP-mannose to exogenous phosphatidic acid or retinyl phosphate. Amphomycin, an inhibitor of dolichyl phosphate mannose synthesis, blocked mannosyl transfer to the endogenous lipid, and to exogenous retinyl phosphate and phosphatidic acid. We conclude that the same mannosyltransferase responsible for dolichyl phosphate mannose synthesis can also utilize in vitro exogenous retinyl phosphate and phosphatidic acid as well as endogenous phosphatidic acid as mannosyl acceptors.

Dolichyl phosphate-mannosyltransferase and dolichyl phosphate-N-acetylglucosaminyltransferase activities in liver preparations from normal controls and patients with cystic fibrosis and diabetes mellitus.

Optimal assay conditions have been determined in human liver preparations for the catalytic transfer of mannose and N-acetylglucosamine from GDP-mannose and UDP-N-acetylglucosamine, respectively, to dolichyl phosphate. Both enzymatic reactions have an absolute requirement for divalent cation (5 mmol/l Mn2+ optimal), detergent (Triton X-100 or Nonidet P-40) and dolichyl phosphate (as acceptor substrate) and both reactions have optimal activity at a pH value of 7.8. Preliminary characterization of the glycolipid products for both enzymatic reactions indicates that phosphorylated dolichol is the major acceptor substrate for radiolabeled mannose and N-acetylglucosamine. The activity levels and specific activities of dolichyl phosphate-mannosyltransferase are comparable in liver homogenates from normal controls and patients with cystic fibrosis and diabetes mellitus. The activity levels and specific activities of dolichyl phosphate-N-acetylglucosaminyltransferase are comparable in liver homogenates from normal controls and patients with cystic fibrosis and diabetes mellitus but considerably lower than the activity levels of dolichyl phosphate-mannosyltransferase. It appears that two of the initial steps of the lipid-mediated glycosylation pathway are normal in livers from patients with cystic fibrosis and diabetes mellitus.