High ribulose-1,5-bisphosphate carboxylase/oxygenase content in northern diatom species

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a fundamental enzyme in CO2-fixation in photoautotrophic organisms. Nonetheless, it has been recently suggested that the contribution of this enzyme to total cellular protein is low in phytoplankton, including diatoms (< 6%). Here we show that RuBisCO content is high in some diatom species isolated from northern waters (> 69°N). Two species contained the highest RuBisCO levels ever reported for phytoplankton (36% of total protein). These high RuBisCO requirements do not increase these species’ requirements for nitrogen and do not impart a fitness disadvantage in terms of growth rate. On the contrary, high RuBisCO levels in psychrophilic diatoms may be a necessary mechanism to maintain high growth rates at low temperature at which enzymatic rates are low.

Estimating the protein burden limit of yeast cells by measuring the expression limits of glycolytic proteins

The ultimate overexpression of a protein could cause growth defects, which are known as the protein burden. However, the expression limit at which the protein-burden effect is triggered is still unclear. To estimate this limit, we systematically measured the overexpression limits of glycolytic proteins in Saccharomyces cerevisiae. The limits of some glycolytic proteins were up to 15% of the total cellular protein. These limits were independent of the proteins’ catalytic activities, a finding that was supported by an in silico analysis. Some proteins had low expression limits that were explained by their localization and metabolic perturbations. The codon usage should be highly optimized to trigger the protein-burden effect, even under strong transcriptional induction. The S–S-bond-connected aggregation mediated by the cysteine residues of a protein might affect its expression limit. Theoretically, only non-harmful proteins could be expressed up to the protein-burden limit. Therefore, we established a framework to distinguish proteins that are harmful and non-harmful upon overexpression.

Stressful preconditioning and HSP70 overexpression attenuate proteotoxicity of cellular ATP depletion

Rat H9c2 myoblasts were preconditioned by heat or metabolic stress followed by recovery under normal conditions. Cells were then subjected to severe ATP depletion, and stress-associated proteotoxicity was assessed on 1) the increase in a Triton X-100-insoluble component of total cellular protein and 2) the rate of inactivation and insolubilization of transfected luciferase with cytoplasmic or nuclear localization. Both heat and metabolic preconditioning elevated the intracellular heat shock protein 70 (HSP70) level and reduced cell death after sustained ATP depletion without affecting the rate and extent of ATP decrease. Each preconditioning attenuated the stress-induced insolubility among total cellular protein as well as the inactivation and insolubilization of cytoplasmic and nuclear luciferase. Transient overexpression of human HSP70 in cells also attenuated both the cytotoxic and proteotoxic effects of ATP depletion. Quercetin, a blocker of stress-responsive HSP expression, abolished the effects of stressful preconditioning but did not influence the effects of overexpressed HSP70. Analyses of the cellular fractions revealed that both the stress-preconditioned and HSP70-overexpressing cells retain the soluble pool of HSP70 longer during ATP depletion. Larger amounts of other proteins coimmunoprecipitated with excess HSP70 compared with control cells deprived of ATP. This is the first demonstration of positive correlation between chaperone activity within cells and their viability in the context of ischemia-like stress.

Novel Method for Rapid Measurement of Growth of Mycobacteria in Detergent-Free Media

We describe a novel, rapid, and inexpensive method for the measurement of growth of Mycobacterium tuberculosis, Mycobacterium bovis, andMycobacterium smegmatis in the presence or absence of detergent. The method, which employs hot NaOH treatment of mycobacterial cells to release total cellular protein, compares favorably with other methods for monitoring mycobacterial growth but is particularly useful for heavily clumped cultures grown in defined minimal medium.

Purification and characterization of histone H1 from meiotic cells of a monocotyledonous plant, Lilium longiflorum

To identify molecules involved in regulating meiotic chromatin structure, nuclear proteins from meiocytes of Lilium longiflorum were chromatographed on hydroxylapatite and the bound and unbound proteins were examined. An abundant nuclear protein was purified from the unbound fraction and by the following criteria was identified as a histone H1 molecule. The protein is soluble in acidic (perchloric and sulfuric acid) solutions, and its amino acid composition and the sequence of its amino terminus are similar to that of known histone H1s. An antiserum was produced against the protein to facilitate subsequent studies. Immunoblotting experiments demonstrated that histone H1 immunostaining declines in the developmental interval spanning the diplotene to tetrads stages. Concommitant with this decline is the appearance of several lower molecular mass, cross-reacting proteins. Neither the identity nor roles of these proteins is known. Immunoblotting experiments also demonstrate that, while the level of the protein is relatively constant in nuclei prepared from meiotic and vegetative cells, histone H1 is apparently enriched in total cellular extracts of meiotic cells compared with vegetative cells. This difference was found to be at least 16-fold. I conclude that in meiotic cells, histone H1 accounts for more of the total cellular protein than it does in vegetative cells. The difference in its relative abundance as a percent of the total cellular protein is probably in part due to differences in the ratio of nuclear to cytoplasmic volume in the different cell types, or the purging of sporophytic proteins from the cytoplasm of the meiocytes, or both.Key words: meiosis, histone H1, immunoblotting, meiotic purging.

Effect of heterologous expression of acyl-CoA-binding protein on acyl-CoA level and composition in yeast

We have expressed a bovine synthetic acyl-CoA-binding protein (ACBP) gene in yeast (Saccharomyces cerevisiae) under the control of the GAL1 promoter. The heterologously expressed bovine ACBP constituted up to 6.4% of total cellular protein and the processing was identical with that of native bovine ACBP, i.e. the initiating methionine was removed and the following serine residue was N-acetylated. The expression of this protein did not affect the growth rate of the cells. Determination of the yeast acyl-CoA pool size showed a close positive correlation between the ACBP content of the cells and the size of the acyl-CoA pool. Thus ACBP can act as an intracellular acyl-CoA pool former. Possible physiological functions of ACBP in cells are discussed.

Constant expression and activity of protein phosphatase 2A in synchronized cells.

The levels of the A, B, and C subunits of protein phosphatase 2A in extracts from synchronized embryonic bovine tracheal cells were determined by immunoblotting with subunit-specific antibodies. A constant amount of each subunit was found in resting cells as well as in growing cells from all stages of the cell cycle. The phosphatase activity of protein phosphatase 2A was also constant. A quantitative comparison showed that the A and C subunits were present in similar amounts, whereas the B subunit was present at a significantly lower level. Together, the A, B, and C subunits represented approximately 0.2% of the total cellular protein.