Walls of the mycelial (M) and yeast-like (Y) forms of Mucor rouxii were prepared with the carbohydrate labelled with 14C and the peptide and polypeptide labelled with SH. Walls were extracted by sequential treatment with EDTA, NaOH and acetic acid. The polymers released were separated by glass-fibre paper electrophoresis and gel filtration which showed the presence, in both M and Y walls, of high molecular weight strongly acidic polysaccharides, high molecular weight weakly acidic glycoproteins and low molecular weight glycopeptides, which are weakly acidic or neutral. The strongly acidic polysaccharides from both M and Y forms contained D-glucuronic acid, D-mannose, D-galactose and L-fucose ; the glycoproteins and glycopeptides from both forms had D-mannose as the major sugar. Y walls gave in addition a weakly acidic polysaccharide containing D-glucuronic acid and D-mannose. The strongly acidic polysaccharides of the M form contained a greater proportion of D-galactose and L-fucose than did corresponding polymers of the Y form. The differences in composition of these polymers are discussed in terms of the possible differences in their structure and the relationship to morphology.
The relationship between genetically different seed sizes and seedling survival under severe nutrient deprivation was determined by comparing ten inbred lines of Arabidopsis thaliana. Seedlings were supplied with only sterile distilled water, and the number of days between germination and death (complete chlorosis) was recorded. Seedlings from genotypes with larger seeds survived longer than seedlings from genotypes with smaller seeds. These results suggest a genetically based adaptive significance of larger seed size resulting from a greater seedling tolerance of nutrient deprivation. This may confer a potentially important selective advantage when nutrient deprivation is the result of a low resource supply in the environment, or the result of nutrient depletion by neighbors.
The Tat (twin-arginine translocation) system is a protein targeting pathway utilized by prokaryotes and chloroplasts. Tat substrates are produced with distinctive N-terminal signal peptides and are translocated as fully folded proteins. In Escherichia coli, Tat-dependent proteins often contain redox cofactors that must be loaded before translocation. Trimethylamine N-oxide reductase (TorA) is a model bacterial Tat substrate and is a molybdenum cofactor-dependent enzyme. Co-ordination of cofactor loading and translocation of TorA is directed by the TorD protein, which is a cytoplasmic chaperone known to interact physically with the TorA signal peptide. In the present study, a pre-export TorAD complex has been characterized using biochemical and biophysical techniques, including SAXS (small-angle X-ray scattering). A stable, cofactor-free TorAD complex was isolated, which revealed a 1:1 binding stoichiometry. Surprisingly, a TorAD complex with similar architecture can be isolated in the complete absence of the 39-residue TorA signal peptide. The present study demonstrates that two high-affinity binding sites for TorD are present on TorA, and that a single TorD protein binds both of those simultaneously. Further characterization suggested that the C-terminal ‘Domain IV’ of TorA remained solvent-exposed in the cofactor-free pre-export TorAD complex. It is possible that correct folding of Domain IV upon cofactor loading is the trigger for TorD release and subsequent export of TorA.
Polyuronides were extracted from purified yeast and mycelial walls of Mucor rouxii by sequential treatments with lithium chloride and potassium hydroxide and were fractionated by ion-exchange chromatography on DEAE-Sephadex. Two polymers (I and II) of different acidity were found in both wall types. Polymer I contained D-glucuronic acid, L-fucose, D-mannose, and much smaller amounts of D-galactose. Yeast and mycelial polymer I had similar uronic acid contents but differed in their neutral sugar compositions and molecular weights. Polymer II from both cell types contained largely D-glucuronic acid and had similar molecular weights. On partial acid hydrolysis, both polymers I and II gave rise to insoluble glucuronans which appeared to be homopolymeric. One-third of the total uronosyl residues of polymer I, and almost all of the uronosyl residues of polymer II, were present in homopolymeric segments. However, homopolymers derived from polymers I and II may not be identical.
Culture filtrates of Mucor rouxii contained oligomers of glucuronic acid which were labeled rapidly during pulses with D-[U-'4C]glucose. These oligomers were probably derived by enzymatic lysis of acidic polymers in the cell wall. The kinetics of the incorporation of label into oligouronides and cell wall polymers suggested that lysis of the wall was required for active hyphal extension. Experiments with cycloheximide, which inhibited hyphal extension, suggested that wall lysis was also required for the subapical cell wall synthesis which probably occurred under these conditions.
Escherichia coli is a Gram‐negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the periplasmic nitrate reductase NapA contains two types of redox cofactor and is exported across the cytoplasmic membrane by the twin‐arginine protein transport pathway. NapD is a small cytoplasmic protein that is essential for the activity of the periplasmic nitrate reductase and binds tightly to the twin‐arginine signal peptide of NapA. Here we show, using spin labelling and EPR, that the isolated twin‐arginine signal peptide of NapA is structured in its unbound form and undergoes a small but significant conformational change upon interaction with NapD. In addition, a complex comprising the full‐length NapA protein and NapD could be isolated by engineering an affinity tag onto NapD only. Analytical ultracentrifugation demonstrated that the two proteins in the NapDA complex were present in a 1 : 1 molar ratio, and small angle X‐ray scattering analysis of the complex indicated that NapA was at least partially folded when bound by its NapD partner. A NapDA complex could not be isolated in the absence of the NapA Tat signal peptide. Taken together, this work indicates that the NapD chaperone binds primarily at the NapA signal peptide in this system and points towards a role for NapD in the insertion of the molybdenum cofactor.Structured digital abstract
NapD and NapA
bind by x ray scattering (View interaction)NapA and NapD physically interact by molecular sieving (View interaction)NapA and NapD
bind by electron paramagnetic resonance (View interaction)
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