beta-subunits of Snf1 kinase are required for kinase function and substrate definition.
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The Snf1 kinase and its mammalian homolog, the AMP-activated protein kinase, are heterotrimeric enzymes composed of a catalytic alpha-subunit, a regulatory gamma-subunit and a beta-subunit that mediates heterotrimer formation. Saccharomyces cerevisiae encodes three beta-subunit genes, SIP1, SIP2 and GAL83. Earlier studies suggested that these subunits may not be required for Snf1 kinase function. We show here that complete and precise deletion of all three beta-subunit genes inactivates the Snf1 kinase. The sip1Delta sip2Delta gal83Delta strain is unable to derepress invertase, grows poorly on alternative carbon sources and fails to direct the phosphorylation of the Mig1 and Sip4 proteins in vivo. The SIP1 sip2Delta gal83Delta strain manifests a subset of Snf phenotypes (Raf(+), Gly(-)) observed in the snf1Delta 10 strain (Raf(-), Gly(-)), suggesting that individual beta-subunits direct the Snf1 kinase to a subset of its targets in vivo. Indeed, deletion of individual beta-subunit genes causes distinct differences in the induction and phosphorylation of Sip4, strongly suggesting that the beta-subunits play an important role in substrate definition. (+info)
Characterization of pRGO1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria.
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The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6, 868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 x 10(6) CFU/microg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10(4) to 10(7) CFU/microg of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially. (+info)
Signal peptidase and oligosaccharyltransferase interact in a sequential and dependent manner within the endoplasmic reticulum.
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We demonstrate that the signal peptides of prepro-alpha-factor and preinvertase must be cleaved before Asn-X-Ser/Thr acceptor tripeptides located near the signal peptides of these precursors can be efficiently glycosylated within the endoplasmic reticulum of the yeast Saccharomyces cerevisiae. The data support a model whereby the interaction of a signal peptide with the membrane prevents an acceptor tripeptide juxtaposed to the signal peptide from accessing the oligosaccharyltransferase active site until the signal peptide is cleaved. (+info)
Vacuolar storage proteins are sorted in the cis-cisternae of the pea cotyledon Golgi apparatus.
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Developing pea cotyledons contain functionally different vacuoles, a protein storage vacuole and a lytic vacuole. Lumenal as well as membrane proteins of the protein storage vacuole exit the Golgi apparatus in dense vesicles rather than in clathrin-coated vesicles (CCVs). Although the sorting receptor for vacuolar hydrolases BP-80 is present in CCVs, it is not detectable in dense vesicles. To localize these different vacuolar sorting events in the Golgi, we have compared the distribution of vacuolar storage proteins and of alpha-TIP, a membrane protein of the protein storage vacuole, with the distribution of the vacuolar sorting receptor BP-80 across the Golgi stack. Analysis of immunogold labeling from cryosections and from high pressure frozen samples has revealed a steep gradient in the distribution of the storage proteins within the Golgi stack. Intense labeling for storage proteins was registered for the cis-cisternae, contrasting with very low labeling for these antigens in the trans-cisternae. The distribution of BP-80 was the reverse, showing a peak in the trans-Golgi network with very low labeling of the cis-cisternae. These results indicate a spatial separation of different vacuolar sorting events in the Golgi apparatus of developing pea cotyledons. (+info)
Sucrose metabolism in plastids.
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The question whether sucrose (Suc) is present inside plastids has been long debated. Low Suc levels were reported to be present inside isolated chloroplasts, but these were argued to be artifacts of the isolation procedures used. We have introduced Suc-metabolizing enzymes in plastids and our experiments suggest substantial Suc entry into plastids. The enzyme levansucrase from Bacillus subtilis efficiently synthesizes fructan from Suc. Targeting of this enzyme to the plastids of tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) plants leads to high-level fructan accumulation in chloroplasts and amyloplasts, respectively. Moreover, introduction of this enzyme in amyloplasts leads to an altered starch structure. Expression of the yeast invertase in potato tuber amyloplasts results in an 80% reduction of total Suc content, showing efficient hydrolysis of Suc by the plastidic invertase. These observations suggest that Suc can enter plastids efficiently and they raise questions as to its function and metabolism in this organelle. (+info)
Molecular cloning and sequence analysis of an endoinulinase gene from Penicillium sp. strain TN-88.
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A genomic DNA segment and cDNAs encoding an extracellular endoinulinase of Penicillium sp. strain TN-88 were cloned and sequenced. Southern blot analysis indicated that the endoinulinase gene (inuC) was present as a single copy in the genome. An open reading frame, consisting of 1,545 bp, was not interrupted by introns, and it encoded a 25 amino acid signal peptide and a 490 amino acid mature protein. The mature protein contained three Cys residues and ten potential N-linked glycosylation sites. Three distinct transcriptional start points were observed at positions -242 (A), -215 (A), and -75 (C) from the start codon. The 5'-noncoding region had a putative TATA box at position -120 (TATATATA) and two contiguous CAAT sequences at -159 to -151. The deduced amino acid sequence showed 72 and 85% identities with those of Aspergillus niger and Penicillium purpurogenum endoinulinase genes, respectively. A neighbor-joining tree showed that fungal endoinulinases form a distinct cluster from other members of the beta-fructofuranosidase superfamily and that they are more closely related to bacterial levanases than to a fungal fructosyltransferase, yeast invertases, or a yeast exoinulinase. (+info)
Tuberization in potato involves a switch from apoplastic to symplastic phloem unloading.
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Phloem unloading was studied in potato plants in real time during the early stages of tuberization using carboxyfluorescein (CF) as a phloem-mobile tracer, and the unloading pattern was compared with autoradiography of tubers that had transported (14)C assimilates. In stolons undergoing extension growth, apoplastic phloem unloading predominated. However, during the first visible signs of tuberization, a transition occurred from apoplastic to symplastic transport, and both CF and (14)C assimilates subsequently followed identical patterns of phloem unloading. It is suggested that the switch to symplastic sucrose unloading may be responsible for the upregulation of several genes involved in sucrose metabolism. A detailed analysis of sugar levels and (14)C sugar partitioning in tuberizing stolons revealed a distinct difference between the apical region of the tuber and the subapical region. Analysis of invertase activity in nontuberizing and tuberizing stolons revealed a marked decline in soluble invertase in the subapical region of swelling stolons, consistent with the switch from apoplastic to symplastic unloading. However, cell wall-bound invertase activity remained high in the apical 1 to 2 mm of tuberizing stolons. Histochemical analysis of potato lines transformed with the promoter of an apoplastic invertase gene (invGE) linked to a reporter gene also revealed discrete gene expression in the apical bud region. Evidence is presented that the apical and lateral tuber buds function as isolated domains with respect to sucrose unloading and metabolism. (+info)
TRAPP I implicated in the specificity of tethering in ER-to-Golgi transport.
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TRAPP is a conserved protein complex required early in the secretory pathway. Here, we report two forms of TRAPP, TRAPP I and TRAPP II, that mediate different transport events. Using chemically pure TRAPP I and COPII vesicles, we have reconstituted vesicle targeting in vitro. The binding of COPII vesicles to TRAPP I is specific, blocked by GTPgammaS, and, surprisingly, does not require other tethering factors. Our findings imply that TRAPP I is the receptor on the Golgi for COPII vesicles. Once the vesicle binds to TRAPP I, the small GTP binding protein Ypt1p is activated and other tethering factors are recruited. (+info)