Myxococcus xanthus protein C is a major spore surface protein.

Fruiting body formation in Myxococcus xanthus involves the aggregation of cells to form mounds and the differentiation of rod-shaped cells into spherical myxospores. The surface of the myxospore is composed of several sodium dodecyl sulfate (SDS)-soluble proteins, the best characterized of which is protein S (Mr, 19,000). We have identified a new major spore surface protein called protein C (Mr, 30,000). Protein C is not present in extracts of vegetative cells but appears in extracts of developing cells by 6 h. Protein C, like protein S, is produced during starvation in liquid medium but is not made during glycerol-induced sporulation. Its synthesis is blocked in certain developmental mutants but not others. When examined by SDS-polyacrylamide gel electrophoresis, two forms of protein C are observed. Protein C is quantitatively released from spores by treatment with 0.1 N NaOH or by boiling in 1% SDS. It is slowly washed from the spore surface in water but is stabilized by the presence of magnesium. Protein C binds to the surface of spores depleted of protein C and protein S. Protein C is a useful new marker for development in M. xanthus because it is developmentally regulated, spore associated, abundant, and easily purified.

Structure, assembly, and secretion of octameric invertase.

Yeast invertase forms a homo-octamer of core glycosylated subunits during assembly in the lumen of the endoplasmic reticulum. This form has been purified from mutant cells (sec18) in which transport of secreted proteins from the endoplasmic reticulum is blocked. No heterologous protein subunits are found in the purified material. Analysis of invertase derived from wild type cells or from mutant cells blocked at subsequent stages in secretion demonstrates that invertase remains a homo-octamer throughout the pathway even though the extent of subunit glycosylation increases. Purified octameric invertase is dissociated into dimer units that reassociate in the presence of polyethylene glycol. Negatively stained preparations show the dissociated enzyme as individual spheres, whereas octameric invertase appears as four associated spheres. Assembly of the octamer in vitro and in vivo is facilitated by the presence of N-linked carbohydrate. Selective release of dimeric glycosylated invertase from intact yeast cells suggests that oligomerization helps retain the enzyme in the periplasmic space.

Nucleotide sequence of the myxobacterial hemagglutinin gene contains four homologous domains.

Myxococcus xanthus, a Gram-negative bacterium, has a complex life cycle that includes fruiting-body formation, a primitive form of multicellular development. Myxobacterial hemagglutinin (MBHA) is a lectin that is induced during the aggregation phase of fruiting-body formation. We have cloned the gene for MBHA and determined its sequence by the dideoxy chain-termination technique. The sequence data show the probable sites for translational initiation and termination and suggest that MBHA does not contain a cleaved leader signal peptide. The DNA sequence shows four strong internal homologies. The deduced amino acid sequence shows that the protein (Mr 27,920) consists of four highly conserved domains each consisting of 67 amino acids. Thus MBHA is physically multivalent in structure, a requirement for all hemagglutinins.

Isolation of glucose-containing high-mannose glycoprotein core oligosaccharides.

The total cell wall mannoprotein has been isolated from a mutant of Saccharomyces cerevisiae that fails to remove the glucose units of the dolichol-linked precursor after transfer of the oligosaccharide to asparagine units in the protein. The oligosaccharides released from this mannoprotein by endoglucosaminidase H digestion show 1H NMR signals assignable to three alpha-linked glucose units as delta 5.52, 5.27, and 5.17, and a comparison with the chemical shifts of reference compounds shows that these signals are consistent with the structure alpha Glc----2 alpha Glc----3 alpha Man----2. This provides a direct confirmation for the structure previously assigned to the lipid-linked precursor. Analysis of the larger oligosaccharides confirms that the presence of the glucose units does not prevent elongation of the alpha 1----6-linked polymannose backbone or addition of alpha 1----3-linked mannose to the core.

Early steps in processing of yeast glycoproteins.

N-linked oligosaccharides have been examined on glycoproteins accumulated in yeast mutants that are blocked at successive stages in the secretory pathway, and in a new mutant, gls1-1, deficient in removal of glucose from N-linked core oligosaccharides, but not blocked in secretion. Oligosaccharides on invertase, a secreted protein, and carboxypeptidase Y, a vacuolar protein, are matured normally in the gls1 mutant but retain three glucoses/carbohydrate chain. The gls1 mutation is recessive and extracts of mutant cells are inactive in release of labeled glucose from core oligosaccharides. The mutant thus lacks glucosidase I activity but could also be deficient in the other core oligosaccharide glucosidase. When transport from the endoplasmic reticulum is blocked in sec18, N-linked oligosaccharides accumulate with a size corresponding to Man8GlcNAc2 when the normal GLS1 allele is present, and Glc3Man8GlcNAc2 in the gls1 mutant. From this we infer that all glucose units are removed prior to glycoprotein transport from the endoplasmic reticulum.

Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole.

Temperature-sensitive secretory mutants (sec) of S. cerevisiae have been used to evaluate the organelles and cellular functions involved in transport of the vacuolar glycoprotein, carboxypeptidase Y (CPY). Others have shown that CPY (61 kd) is synthesized as an inactive proenzyme (69 kd) that is matured by cleavage of an 8 kd amino-terminal propeptide. sec mutants that are blocked in either of two early stages in the secretory process and accumulate endoplasmic reticulum or Golgi bodies also accumulate precursor forms of CPY when cells are incubated at the nonpermissive temperature (37 degrees C). These forms are converted to a proper size when cells are returned to a permissive temperature (25 degrees C). Vacuoles isolated from sec mutant cells do not contain the proCPY produced at 37 degrees C. These results suggest that vacuolar and secretory glycoproteins require the same cellular functions for transport from the endoplasmic reticulum and from the Golgi body. The Golgi body represents a branch point in the pathway: from this organelle, vacuolar proenzymes are transported to the vacuole for proteolytic processing and secretory proteins are packaged into vesicles.

Compartmentalized assembly of oligosaccharides on exported glycoproteins in yeast.

Temperature-sensitive secretory mutants (sec) of S. cerevisiae have been used to evaluate the stages and localization of glycoprotein oligosaccharide synthesis. At the nonpermissive growth temperature (37 degrees C), the sec mutants accumulate secretory organelles and glycoproteins. Histochemical staining and thin-section electron microscopy reveal that the secreted glycoprotein, acid phosphatase, is contained within one of three distinct organelles that accumulates in different mutants: ER; Golgi-like structures called Berkeley bodies; and 80--100 nm vesicles. When produced at 37 degrees C, invertase and acid phosphatase have less carbohydrate in the mutants that accumulate ER than in other mutants, or than in the wild-type strain. External invertase migrates on SDS-polyacrylamide gels as a heterogeneous species with an apparent molecular weight of 100 to 140 kd. Radiolabeled invertase, immunoprecipitated from extracts of ER-accumulating mutant cells, migrates as a set of three discrete protein species with apparent molecular weights of 79, 81, and 83 kd; the other mutants produce a form more like the secreted enzyme. In each case, removal of N-glycosidically linked oligosaccharides by treatment with endoglycosidase H produces a discrete species that migrates as a protein of 61 kd. Immunochemical analysis of bulk glycoprotein accumulated in the mutants suggests that a major portion of the N-linked oligosaccharide, the outer chain, is added after material passes from the ER.

Genetic analysis of Escherichia coli mutants defective in adenylate kinase and sn-glycerol 3-phosphate acyltransferase.

Complementation analysis with independently isolated plA and adk (adenylate kinase) mutants of Escherichia coli showed that all the mutants belong to the same complementation group. The results suggest that the adk (plsA) locus is the structural gene for adenylate kinase.

Nitrogen control of Salmonella typhimurium: co-regulation of synthesis of glutamine synthetase and amino acid transport systems.

Nitrogen control in Salmonella typhimurium is not limited to glutamine synthetase but affects, in addition, transport systems for histidine, glutamine, lysine-arginine-ornithine, and glutamate-aspartate. Synthesis of both glutamine synthetase and transport proteins is elevated by limitation of nitrogen in the growth medium or as a result of nitrogen (N)-regulatory mutations. Increases in the amounts of these proteins were demonstrated by direct measurements of their activities, by immunological techniques, and by visual inspection of cell fractions after gel electrophoresis. The N-regulatory mutations are closely linked on the chromosome to the structural gene for glutamine synthetase, glnA: we discuss the possibility that they lie in a regulatory gene, glnR, which is distinct from glnA. Increases in amino acid transport in N-regulatory mutant strains were indicated by increased activity in direct transport assays, improved growth on substrates of the transport systems, and increased sensitivity to inhibitory analogs that are trnasported by these systems. Mutations to loss of function of individual transport components (hisJ, hisP, glnH, argT) were introduced into N-regulatory mutant strains to determine the roles of these components in the phenotype and transport behavior of the strains. The structural gene for the periplasmic glutamine-binding protein, glnH, was identified, as was a gene argT that probably encodes the structure of the lysine-arginine-ornithine-binding protein. Genes encoding the structures of the histidine- and glutamine-binding proteins are not linked to glnA or to each other by P22-mediated transduction; thus, nitrogen control is exerted on several unlinked genes.