Effect of heat shock on protein degradation in mammalian cells: involvement of the ubiquitin system.

Exposure of cultured rat hepatoma (HTC) cells to a 43 degrees C heat shock transiently accelerates the degradation of the long-lived fraction of cellular proteins. The rapid phase of proteolysis which lasts approximately 2 h after temperature step-up is followed by a slower phase of proteolysis. During the first 2 h after temperature step-up there is a wave of ubiquitin conjugation to cellular proteins which is accompanied by a fall in ubiquitin and ubiquitinated histone 2A (uH2A) levels. Upon continued incubation at 43 degrees C the levels of ubiquitin conjugates fall with a corresponding increase of ubiquitin and uH2A to initial levels. The burst of protein degradation and ubiquitin conjugation after temperature step-up is not affected by the inhibition of heat shock protein synthesis. Cells of the FM3A ts85 mutant, which have a thermolabile ubiquitin activating enzyme (E1), do not accelerate protein degradation in response to a 43 degrees C heat shock, whereas wild-type FM3A mouse cells do. This observation indicates that the ubiquitin system is involved in the degradation of heat-denatured proteins. Sequential temperature jump experiments show that the extent of proteolysis at temperatures up to 43 degrees C is related to the final temperature and not to the number of steps taken to attain it. Temperature step-up to 45 degrees C causes the inhibition of intracellular proteolysis. We propose the following explanation of the above observations. Heat shock causes the conformational change or denaturation of a subset of proteins stable at normal temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure of the incompatibility factors of Schizophyllum commune: Constitution of the three classes of B factors

Summary The B factors of Schizophyllum commune are of 3 classes: The high recombining class I has 7 alleles and 7 alleles; the low recombining classes are class II with 7 allels and probably 2 alleles and class III with probably 2 (or also 2) alleles and 7 allels. A fourth hypothetical class (-) was not found and either does not exist or is indistinguishable from class III by the tests employed. The and alleles differ from and by either (a) mutations affecting both mating specificity and recombination frequency, or (b) deletions involving most of the B region.The research was supported by a grant from the Atomic Energy Commission of the U.S. No. (30-1)-3875 and was performed at the Biological Laboratories, Harvard University, Cambridge, Mass., U.S.A.     

Stable assembly of PsaE into cyanobacterial photosynthetic membranes is dependent on the presence of other accessory subunits of photosystem I

We studied assembly of the PsaE subunit of photosystem I into photosynthetic membranes of cyanobacterial mutant strains that lack specific photosystem I subunits. Radiolabeled PsaE was incubated with photosynthetic membranes, and their binding and assembly were assayed by resistance to removal by chaotropic agents and proteolytic digestion. PsaE incorporated into the wild-type membranes was resistant to these treatments. In the absence of PsaD, it was resistant to proteolytic digestion, but was removed by NaBr. When the membranes were isolated from a mutant strain in which the psaF and psaJ genes have been inactivated, PsaE assembled in vitro could not be removed. PsaE could associate with the membranes of the strain DF in which the psaD, psaJ and psaF genes have been mutated. However, the radiolabeled PsaE associated with these membranes was removed both by the proteolytic as well as by the chaotropic agents. Characterization of PsaE present in vivo revealed similar results. These observations suggest that PsaD and PsaF/J may interact with PsaE and stabilize it in the photosystem I complex.     

The effects of calcium deficiency on Cucurbita pepo L. hypocotyl cells: A 31P nuclear-magnetic-resonance study

Calcium deficiency in zucchini (Cucurbita pepo L.) is associated with reduced growth and a reduced ability to transport auxin (Allan and Rubery, 1991, Planta 183, 604–612). An investigation of the effects of calcium-deficiency on zucchini hypocotyl cells was made using weak-acid uptake and ³¹P-nuclear-magneticresonance (³¹P-NMR) spectroscopy in vivo and in tissue extracts. Calcium-deficient tissue had the same cytoplasmic and vacuolar pHs as normal tissue when extracellular pH was near neutral. At acidic external pH the vacuolar pH was lower in deficient tissue. Adenine nucleotides were present predominantly as ATP in both control and calcium-deficient tissues. Addition of calcium to calcium-deficient tissue, under conditions which cause recovery of auxin transport induced no changes in the ³¹P-NMR spectra of deficient tissue. The content of mobile, phosphorylated metabolites was reduced in calcium-deficient tissue in comparison to control tissue. However, a substantial increase in the content of phosphorylcholine occurs in calcium-deficient tissues compared with controls; this may reflect changes in lipid turnover in calcium-stressed cells. We wish to thank Drs. Terry Moore and Jamie Vandenberg for technical assistance and Professor Peter Morris for providing the gated oxygen device. A.C.A. thanks the Cambridge Commonwealth Trust for a Prince of Wales Scholarship and the O.R.S. Awards Scheme for an award.     

CAENORHABDITIS ELEGANS Fertilization-Defective Mutants with Abnormal Sperm

Seven new fertilization-defective mutants of C. elegans have been isolated and characterized; six are temperature sensitive, one is absolute and all are autosomal recessive. One mutation is in a previously described gene, while the other six define six new fer genes that appear to code for sperm-specific functions necessary for normal fertilization. In all fer mutants, both males and hermaphrodites accumulate sperm in near normal numbers. In hermaphrodites, mutant sperm contact the oocytes, but fail to fertilize them. Instead, the sperm are swept into the uterus by the passing oocytes and are expelled when oocytes are laid. Males of two fer mutants do not transfer sperm during copulation, but the other mutant males transfer sperm that fail to move to the spermatheca. Spermatozoa from fer-1 and fer-4 mutants are motility-defective in vitro as well as in vivo, and their pseudopods have an altered morphology. The period of development during which mutant hermaphrodites are temperature sensitive for fertility overlaps the time of sperm development. Some mutants are temperature sensitive throughout the entire period, and others are temperature sensitive during or just prior to spermiogenesis. In fer-4/+ and fer-7/+ males, the fertility of the mutation-bearing sperm is diminished, reducing the transmission ratio. This implies some post-meiotic expression of these genes.—This set of mutants provides a variety of functional and structural alterations in nematode sperm that should help identify and analyze gene products involved in sperm morphogenesis and motility.     

Correlation between changes in the membrane organization and susceptibility to phospholipase C attack induced by ATP depletion of rat erythrocytes

About 20 and 43% of the total membrane phospholipids are hydrolized in fresh rat erythrocytes by treatment with phospholipase C (Bacillus cereus), or both sphingomyelinase and phospholipase C, respectively, without causing cell lysis. Treatment of ATP-depleted cells with phospholipase C alone results in 50% hydrolysis and extensive lysis. Depletion of ATP causes a marked increase in the aggregation of intramembranous particles accompanied by a similar increase in the smooth area between the particle clusters as revealed by the freeze-etch technique. Such changes are not induced by extensive phospholipid hydrolysis in absence of cell lysis in fresh cells.Based on these and additional data, it is suggested that the membrane phospholipid organization can be divided into 3 types: phospholipids exposed to phospholipase C; phospholipids protected against phospholipase C by presence of sphingomyelin; phospholipids which can be exposed following alteration of the proteinlipid interactions. Such alterations which might be induced by a variety of means, including ATP depletion, might result in clustering of intramembranous particles and increase of the free lipid bilayer phase of the membrane.     

A high-confidence human plasma proteome reference set with estimated concentrations in PeptideAtlas

Human blood plasma can be obtained relatively noninvasively and contains proteins from most, if not all, tissues of the body. Therefore, an extensive, quantitative catalog of plasma proteins is an important starting point for the discovery of disease biomarkers. In 2005, we showed that different proteomics measurements using different sample preparation and analysis techniques identify significantly different sets of proteins, and that a comprehensive plasma proteome can be compiled only by combining data from many different experiments. Applying advanced computational methods developed for the analysis and integration of very large and diverse data sets generated by tandem MS measurements of tryptic peptides, we have now compiled a high-confidence human plasma proteome reference set with well over twice the identified proteins of previous high-confidence sets. It includes a hierarchy of protein identifications at different levels of redundancy following a clearly defined scheme, which we propose as a standard that can be applied to any proteomics data set to facilitate cross-proteome analyses. Further, to aid in development of blood-based diagnostics using techniques such as selected reaction monitoring, we provide a rough estimate of protein concentrations using spectral counting. We identified 20,433 distinct peptides, from which we inferred a highly nonredundant set of 1929 protein sequences at a false discovery rate of 1%. We have made this resource available via PeptideAtlas, a large, multiorganism, publicly accessible compendium of peptides identified in tandem MS experiments conducted by laboratories around the world.