RNAi knockdown of parafusin inhibits the secretory pathway

Several glycolytic enzymes and their isoforms have been found to be important in cell signaling unrelated to glycolysis. The involvement of parafusin (PFUS), a member of the phosphoglucomutase (PGM) superfamily with no phosphoglucomutase activity, in Ca(2+)-dependent exocytosis has been controversial. This protein was first described in Paramecium tetraurelia, but is widely found. Earlier work showed that parafusin is a secretory vesicle scaffold component with unusual post-translational modifications (cyclic phosphorylation and phosphoglucosylation) coupled to stages in the exocytic process. Using RNAi, we demonstrate that parafusin synthesis can be reversibly blocked, with minor or no effect on other PGM isoforms. PFUS knockdown produces an inhibition of dense core secretory vesicle (DCSV) synthesis leading to an exo(-) phenotype. Although cell growth is unaffected, vesicle content is not packaged properly and no new DCSVs are formed. We conclude that PFUS and its orthologs are necessary for proper scaffold maturation. Because of this association, parafusin is an important signaling component for regulatory control of the secretory pathway.     

Functional diversity of the phosphoglucomutase superfamily: structural implications.

Three-dimensional structural models of three members of the phosphoglucomutase (PGM) superfamily, parafusin, phosphoglucomutase-related protein and sarcoplasmic reticulum phosphoglucomutase, were constructed by homology modeling based on the known crystal structure of rabbit muscle phosphoglucomutase. Parafusin, phosphoglucomutase-related protein and sarcoplasmic reticulum phosphoglucomutase each have 50% or more identity with rabbit muscle phosphoglucomutase at the amino acid level and all are reported to exhibit no or minor phosphoglucomutase activity. There are four major insertions and two deletions in the parafusin sequence relative to PGM, all of which are located in surface-exposed loops connecting secondary structural elements. The remaining amino acid substitutions are distributed throughout the sequence and are not predicted to alter the polypeptide fold. Parafusin contains a putative protein kinase C site located on a surface loop in domain II that is not present in the homologs. Although the general domain structure and the active site of rabbit muscle phosphoglucomutase are preserved in the model of phosphoglucomutase-related protein, a major structural difference is likely to occur in domain 1 due to the absence of 55 amino acid residues in PGM-RP. This deletion predicts the loss of three alpha-helices and one beta-strand from an anti-parallel beta-sheet in this domain as compared with the rabbit muscle phosphoglucomutase.     

Molecular identification of mammalian phosphopentomutase and glucose-1,6-bisphosphate synthase, two members of the alpha-D-phosphohexomutase family

The molecular identity of mammalian phosphopentomutase has not yet been established unequivocally. That of glucose-1,6-bisphosphate synthase, the enzyme that synthesizes a cofactor for phosphomutases and putative regulator of glycolysis, is completely unknown. In the present work, we have purified phosphopentomutase from human erythrocytes and found it to copurify with a 68-kDa polypeptide that was identified by mass spectrometry as phosphoglucomutase 2 (PGM2), a protein of the alpha-d-phosphohexomutase family and sharing about 20% identity with mammalian phosphoglucomutase 1. Data base searches indicated that vertebrate genomes contained, in addition to PGM2, a homologue (PGM2L1, for PGM2-like 1) sharing about 60% sequence identity with this protein. Both PGM2 and PGM2L1 were overexpressed in Escherichia coli, purified, and their properties were studied. Using catalytic efficiency as a criterion, PGM2 acted more than 10-fold better as a phosphopentomutase (both on deoxyribose 1-phosphate and on ribose 1-phosphate) than as a phosphoglucomutase. PGM2L1 showed only low (<5%) phosphopentomutase and phosphoglucomutase activities compared with PGM2, but was about 5-20-fold better than the latter enzyme in catalyzing the 1,3-bisphosphoglycerate-dependent synthesis of glucose 1,6-bisphosphate and other aldose-bisphosphates. Furthermore, quantitative real-time PCR analysis indicated that PGM2L1 was mainly expressed in brain where glucose-1,6-bisphosphate synthase activity was previously shown to be particularly high. We conclude that mammalian phosphopentomutase and glucose-1,6-bisphosphate synthase correspond to two closely related proteins, PGM2 and PGM2L1, encoded by two genes that separated early in vertebrate evolution.     

Evolutionary implications of localization of the signaling scaffold protein Parafusin to both cilia and the nucleus

Parafusin (PFUS), a 63 kDa protein first discovered in the eukaryote Paramecium and known for its role in apicomplexan exocytosis, provides a model for the common origin of cellular systems employing scaffold proteins for targeting and signaling. PFUS is closely related to eubacterial rather than archeal phosphoglucomutases (PGM) – as we proved by comparison of their 88 sequences – but has no PGM activity. Immunofluorescence microscopy analysis with a PFUS‐specific peptide antibody showed presence of this protein around the base region of primary cilia in a variety of mammalian cell types, including mouse embryonic (MEFs) and human foreskin fibroblasts (hFFs), human carcinoma stem cells (NT‐2 cells), and human retinal pigment epithelial (RPE) cells. Further, PFUS localized to the nucleus of fibroblasts, and prominently to nucleoli of MEFs. Localization studies were confirmed by Western blot analysis, showing that the PFUS antibody specifically recognizes a single protein of ca. 63 kDa in both cytoplasmic and nuclear fractions. Finally, immunofluorescence microscopy analysis showed that PFUS localized to nuclei and cilia in Paramecium. These results support the suggestion that PFUS plays a role in signaling between nucleus and cilia, and that the cilium and the nucleus both evolved around the time of eukaryotic emergence. We hypothesize that near the beginnings of eukaryotic cell evolution, scaffold proteins such as PFUS arose as peripheral membrane protein identifiers for cytoplasmic membrane trafficking and were employed similarly during the subsequent evolution of exocytic, nuclear transport, and ciliogenic mechanisms.     

A phylogenetic approach to the identification of phosphoglucomutase genes

The expanding molecular database provides unparalleled opportunities for characterizing genes and for studying groups of related genes. We use sequences drawn from the database to construct an evolutionary framework for examining the important glycolytic enzyme phosphoglucomutase (PGM). Phosphoglucomutase plays a pivotal role in the synthesis and utilization of glycogen and is present in all organisms. In humans, there are three well-described isozymes, PGMI, PGM2, and PGM3. PGM1 was cloned 5 years ago; however, repeated attempts using both immunological approaches and molecular probes designed from PGM1 have failed to isolate either PGM2 or PGM3. Using a phylogenetic strategy, we first identified 47 highly divergent prokaryotic and eukaryotic PGM-like sequences from the database. Although overall amino acid identity often fell below 20%, the relative order, position, and sequence of three structural motifs, the active site and the magnesium-- and sugar-binding sites, were conserved in all 47 sequences. The phylogenetic history of these sequences was complex and marked by duplications and translocations; two instances of transkingdom horizontal gene transfer were identified. Nonetheless, the sequences fell within six well-defined evolutionary lineages, three of which contained only prokaryotes. Of the two prokaryotic/eukaryotic lineages, one contained bacterial, yeast, slimemold, invertebrate, and vertebrate homologs to human PGM1 and the second contained likely homologs to human PGM2. Indeed, an amino acid sequence, derived from a partial human cDNA, that fell within the second cross-kingdom lineage bears several characteristics expected for PGM2. A third lineage may contain homologs to human PGM3. On a general level, our phylogenetic-based approach shows promise for the further utilization of the extensive molecular database.      

A parafusin-related Toxoplasma protein in Ca2+-regulated secretory organelles

We cloned a gene, PRPI, of Toxoplasma gondii encoding a 637-amino-acids protein having a calculated mass of 70 kDa. The sequence showed high homology to parafusin, a protein that in Paramecium tetraurelia participates in Ca2+-regulated exocytosis and is a paralog of phosphoglucomutase. We show that Toxoplasma gondii homogenate and an expressed recombinant PRP1 fusion protein cross-react with a specific peptide-derived antibody to parafusin in Western blots. Antibodies to the recombinant PRP1 showed cross-reaction with parafusin and recognized PRP1, as bands at M, 63 x 10(3) and 68 x 10(3), respectively. PRP1 is labeled when Toxoplasma gondii cells are incubated with inorganic 32P and appears as the major band on autoradiograms of SDS-PAGE gels. The localization of PRP1 was examined in secretory organelles of Toxoplasma gondii by deconvolution light microscopy followed by three dimensional reconstruction using pairwise combinations of specific antibodies. PRP1 localized to the apical third of the cell. It co-localized with micronemes, the only secretory organelle the secretion of which is Ca2+ dependent. Quantification of the co-localized stain suggests that only mature micronemes ready for exocytosis have PRP1. These findings suggest that PRP1, parafusin and other members of the phosphoglucomutase superfamily have a conserved role in Ca2+-regulated exocytic processes.     

Mammalian phosphomannomutase PMM1 is the brain IMP-sensitive glucose-1,6-bisphosphatase

Glucose 1,6-bisphosphate (Glc-1,6-P(2)) concentration in brain is much higher than what is required for the functioning of phosphoglucomutase, suggesting that this compound has a role other than as a cofactor of phosphomutases. In cell-free systems, Glc-1,6-P(2) is formed from 1,3-bisphosphoglycerate and Glc-6-P by two related enzymes: PGM2L1 (phosphoglucomutase 2-like 1) and, to a lesser extent, PGM2 (phosphoglucomutase 2). It is hydrolyzed by the IMP-stimulated brain Glc-1,6-bisphosphatase of still unknown identity. Our aim was to test whether Glc-1,6-bisphosphatase corresponds to the phosphomannomutase PMM1, an enzyme of mysterious physiological function sharing several properties with Glc-1,6-bisphosphatase. We show that IMP, but not other nucleotides, stimulated by >100-fold (K(a) approximately 20 mum) the intrinsic Glc-1,6-bisphosphatase activity of recombinant PMM1 while inhibiting its phosphoglucomutase activity. No such effects were observed with PMM2, an enzyme paralogous to PMM1 that physiologically acts as a phosphomannomutase in mammals. Transfection of HEK293T cells with PGM2L1, but not the related enzyme PGM2, caused an approximately 20-fold increase in the concentration of Glc-1,6-P(2). Transfection with PMM1 caused a profound decrease (>5-fold) in Glc-1,6-P(2) in cells that were or were not cotransfected with PGM2L1. Furthermore, the concentration of Glc-1,6-P(2) in wild-type mouse brain decreased with time after ischemia, whereas it did not change in PMM1-deficient mouse brain. Taken together, these data show that PMM1 corresponds to the IMP-stimulated Glc-1,6-bisphosphatase and that this enzyme is responsible for the degradation of Glc-1,6-P(2) in brain. In addition, the role of PGM2L1 as the enzyme responsible for the synthesis of the elevated concentrations of Glc-1,6-P(2) in brain is established.     

Role of phosphoglucomutase in lipooligosaccharide biosynthesis in Neisseria gonorrhoeae.

A region of pSG30 that complements the pyocin-derived gonococcal lipooligosaccharide (LOS) mutants 1291d and 1291e was characterized by DNA sequence analysis and an open reading frame of 1,380 bases was identified that is 89% similar and 56% identical over 452 amino acids to the algC gene product from Pseudomonas aeruginosa that encodes phosphomannomutase. Enzymatic analysis of gonococcal crude protein extracts demonstrated that pSG30 encodes phosphoglucomutase (PGM) and phosphomannomutase activity. This activity is absent in 1291d and 1291e but is restored upon introduction of pSG30. PGM encoded by pSG34, a subclone of pSG30, was able to complement Escherichia coli PGM1, a strain deficient in PGM, as determined by bacteriophage C21 plaque formation. A revertant of 1291d that binds monoclonal antibody 2-1-L8 (specific for a 3.6-kDa LOS component) was isolated. The construction of a site-specific deletion of this region in the chromosome of 1291 confirms the role of this open reading frame in LOS biosynthesis.     

Comparison of in vivo and in vitro phosphorylation of the exocytosis-sensitive protein PP63/parafusin by differential MALDI mass spectrometric peptide mapping.

PP63 (parafusin) is a 63 kDa phosphoprotein, which exists in at least two different isoforms. It is very rapidly (80 ms) dephosphorylated during triggered trichocyst exocytosis. This occurs selectively in exocytosis-competent Paramecium tetraurelia strains. At least two protein kinases isolated from Paramecium, casein kinase type II kinase and cGMP-dependent kinase, are able to phosphorylate the two recombinant PP63/parafusin isoforms, both with phosphoglucomutase activity, in vitro. By performing mass spectrometric peptide mapping, we have investigated in vitro phosphorylation of recombinant PP63/parafusin by these kinases in comparison to in vivo phosphorylation of native PP63/parafusin isolated from Paramecium homogenates. Low picomolar quantities of proteolytic digests of recombinant and native PP63/parafusin, prior to and following alkaline phosphatase treatment, were directly analyzed by MALDI mass spectrometry. In native PP63-1/parafusin-1, six of 64 serine and threonine residues (S-196, T-205, T-280, T-371, T-373, and T-469) were found definitely, 27 were found possibly phosphorylated, 28 were identified as nonphosphorylated, and three were not covered by mapping. Three of the six certainly phosphorylated amino acids represent consensus phosphorylation sites for casein kinase II or cGMP-dependent protein kinase. In vitro phosphorylation studies of recombinant PP63/parafusin confirm that some of the sites found were used in vivo; however, also significant differences with respect to in vivo phosphorylation of native PP63/parafusin were observed. The two Paramecium protein kinases that were used do not preferably phosphorylate expected consensus sites in vitro. Homology structure modeling of PP63/parafusin with rabbit phosphoglucomutase revealed that the majority of residues found phosphorylated is located on the surface of the molecule.     

Parafusin and calcium-induced signal transduction in exocytosis

Summary The phosphoglycoprotein parafusin is a member of the phosphoglucomutase superfamily and has been shown, both via biochemical and localization studies, to be associated with the Ca²⁺-dependent regulated exocytosis process inParamecium tetraurelia. Stimulation of exocytosis in this cell leads to a Ca²⁺-dependent glucosylation of parafusin accompanied by its dissociation from the secretory vesicles and from cell membrane docking sites. These events are blocked in the presence of extracellular Mg²⁺ in wild-type cells and in either Ca²⁺ or Mg²⁺ in a temperature-sensitive mutant, nd9, stimulated at the nonpermissive temperature. Furthermore, laser scanning confocal localization studies with antibodies to parafusin whole protein versus antibody made to a specific peptide (insertion 2) show different localization patterns. While insertion-2 antibodies only label the organelles previously shown to have parafusin associated with them, i.e., cell membrane fusion (docking) sites and secretory vesicles, antibodies to whole protein outline in addition the alveolar sacs (subsurface cisterns) which are Ca²⁺ storage compartments in this cell. This may indicate tht other members of the phosphoglucomutase superfamily which interact specifically with this compartment are present inP. tetraurelia.     

Isoenzymes of phosphoglucomutase from human red blood cells: isolation and kinetic properties

A procedure has been developed for the purification of phosphoglucomutase from human red cell (phenotype PGM1 a1 or a3) lysates. It yields homogeneous isoenzyme preparations of the products ("primary" and "secondary") of the two PGM1 and PGM2 loci with distinctive pI (from 6.07 to 5.29). There are substantial differences between PGM1 and PGM2 isoenzymes, having single polypeptide chains of 58,500 and 69,000 Mr respectively and showing different thermostability. The kinetic properties of all the isoenzymes for the phosphoglucomutase reaction are essentially the same (apart from the specific activity of 1089-1263 units/mg for PGM1 forms vs 37-42 units/mg for PGM2 forms), but there are striking differences in substrate specificity. In fact the products of PGM1 locus are "true" phosphoglucomutases, being specific to mutate glucose monophosphates, whereas the PGM2 forms also display phosphoribomutase and glucose 1,6-bisphosphate synthetic activities. Some kinetic properties of these "side activities" are also reported.     

In Vivo Analysis of the Major Exocytosis-sensitive Phosphoprotein in Tetrahymena

Phosphoglucomutase (PGM) is a ubiquitous highly conserved enzyme involved in carbohydrate metabolism. A number of recently discovered PGM-like proteins in a variety of organisms have been proposed to function in processes other than metabolism. In addition, sequence analysis suggests that several of these may lack PGM enzymatic activity. The best studied PGM-like protein is parafusin, a major phosphoprotein in the ciliate Paramecium tetraurelia that undergoes rapid and massive dephosphorylation when cells undergo synchronous exocytosis of their dense-core secretory granules. Indirect genetic and biochemical evidence also supports a role in regulated exocytotic membrane fusion. To examine this matter directly, we have identified and cloned the parafusin homologue in Tetrahymena thermophila, a ciliate in which protein function can be studied in vivo. The unique T. thermophila gene, called PGM1, encodes a protein that is closely related to parafusin by sequence and by characteristic post-translational modifications. Comparison of deduced protein sequences, taking advantage of the known atomic structure of rabbit muscle PGM, suggests that both ciliate enzymes and all other PGM-like proteins have PGM activity. We evaluated the activity and function of PGM1 through gene disruption. Surprisingly, ΔPGM1 cells displayed no detectable defect in exocytosis, but showed a dramatic decrease in PGM activity. Both our results, and reinterpretation of previous data, suggest that any potential role for PGM-like proteins in regulated exocytosis is unlikely to precede membrane fusion.     

The posttranslational modification of phosphoglucomutase is regulated by galactose induction and glucose repression in Saccharomyces cerevisiae.

The enzyme phosphoglucomutase functions at a key point in carbohydrate metabolism. In this paper, we show that the synthesis of the major isoform of yeast phosphoglucomutase, encoded by the GAL5 (PGM2) gene, is regulated in a manner that is distinct from that previously described for other enzymes involved in galactose metabolism in the yeast Saccharomyces cerevisiae. Accumulation of this isoform increased four- to sixfold when the culture experienced either glucose depletion or heat shock. However, heat shock induction did not occur unless the cells were under glucose repression. This nonadditive increase in expression suggests that the regulatory mechanisms controlling the heat shock induction and glucose repression of the GAL5 gene are functionally related. We previously demonstrated that phosphoglucomutase is modified by a posttranslational Glc-phosphorylation reaction. We now show that this posttranslational modification, like phosphoglucomutase expression itself, is also regulated by galactose induction and glucose repression. Finally, no evidence was found to indicate that the Glc-phosphorylation of phosphoglucomutase alters its enzymatic activity under the conditions examined.     

Isolation of the human sex determining region from a Y-enriched yeast artificial chromosome library

We describe the isolation and characterization of yeast artificial chromosome (YAC) clones spanning the male sex determining region on the short arm of the human Y chromosome. The clones were isolated by hybridizing probes in the interval between the genes MIC2 and ZFY to a Y chromosome-enriched YAC library. The YAC clones were consistent with the order of probes established for this interval and may be useful for functional studies of the region in male sex determination. However, many of the YAC clones from this library carried only one arm of the vector ("half-YACs"), deleted sequences from one end, and contained much smaller inserts (148 kb average) than the size of ligated fragments selected by pulsed-field gel electrophoresis (greater than 440 kb). These problems were overcome by protecting DNA with polyamines during YAC library construction and a second Y-enriched YAC library was constructed with an average insert size of 627 kb.