PubMed Health⌕ Search

Biomedical subjects

Y Shu

Publications and source records attributed to Y Shu.

63 records · Page 4Linked to original sources
PubMed1995

Limitations to in vivo import of hydrophobic proteins into yeast mitochondria. The case of a cytoplasmically synthesized apocytochrome b.

The apocytochrome b gene, exclusively encoded by the mitochondrial genome, was engineered so that it could be expressed in the yeast cytoplasm. Different combinations of the apocytochrome b transmembrane domains were produced in the form of hybrid proteins fused to both the N-terminal mitochondrial targeting sequence of the ATPase subunit 9 from Neurospora crassa and to a cytoplasmic version of the bI4 RNA maturase, localised on the N-terminal and C-terminal sides, respectively, of the hydrophobic stretches. The bI4 RNA maturase, which can complement mitochondrial mutations, was used as an in vivo reporter to assess the mitochondrial import of the different groups of transmembrane helices. This new, reliable and sensitive reporter activity allowed us to experimentally determine the limitations to the mitochondrial import of hydrophobic proteins. All eight transmembrane helices of apocytochrome b could be imported into mitochondria, either alone or in combination, but no more than three to four transmembrane helices could be imported together at one time. This limit is close to that observed in the population of nuclear-encoded mitochondrial proteins. The hydrophobic characteristics of engineered and natural proteins targeted to the mitochondrial inner membrane revealed two factors important in the import process. These were (a) the local hydrophobicity of a transmembrane segment, and (b) the average regional hydrophobicity of the protein over an extended length of 60-80 residues. Such features may have played a major role in the evolution of mitochondrial genomes.

Amino Acid Sequence↗
PubMed1995

SCS1, a multicopy suppressor of hsp60-ts mutant alleles, does not encode a mitochondrially targeted protein.

We identified and isolated a Saccharomyces cerevisiae gene which, when overexpressed, suppressed the temperature-sensitive phenotype of cells expressing a mutant allele of the gene encoding the mitochondrial chaperonin, Hsp60. This gene, SCS1 (suppressor of chaperonin sixty-1), encodes a 757-amino-acid protein of as yet unknown function which, nonetheless, has human, rice, and Caenorhabditis elegans homologs with high degrees (ca. 60%) of amino acid sequence identity. SCS1 is not an essential gene, but SCS1-null strains do not grow above 37 degrees C and show some growth-related defects at 30 degrees C as well. This gene is expressed at both 30 and 38 degrees C, producing little or no differences in mRNA levels at these two temperatures. Overexpression of SCS1 could not complement an HSP60-null allele, indicating that suppression was not due to the bypassing of Hsp60 activity. Of 10 other hsp60-ts alleles tested, five could also be suppressed by SCS1 overexpression. There were no common mutant phenotypes of the strains expressing these alleles that give any clue as to why they were suppressible while others were not. An epitope (influenza virus hemagglutinin)-tagged form of SCS1 in single copy complemented an SCS1-null allele. The Scs1-hemagglutinin protein was found to be at comparable levels and in similar multiply modified forms in cells growing at both 30 and 38 degrees C. Surprisingly, when localized either by cell fractionation procedures or by immunocytochemistry, these proteins were found not in mitochondria but in the cytosol. The overexpression of SCS1 had significant effects on the cellular levels of mRNAs encoding the proteins Cpn10 and Mgel, two other mitochondrial protein cochaperones, but not on mRNAs encoding a number of other mitochondrial or cytosolic proteins analyzed. The implications of these findings are discussed.

Alleles↗
PubMed1994

Influence of exercise training on myocardial beta-adrenergic signal transduction: differential regulation with age.

Exercise training is known to increase cardiovascular performance and decrease heart rate. Because activation of adenylyl cyclase is an important factor in beta-adrenergic signal transduction and in the decline in signal transduction with age, we hypothesized that some of the effects of exercise training may be mediated by changes in postreceptor activation of adenylyl cyclase. To this end, we assessed isoproterenol-, G protein-, and forskolin-mediated activation of adenylyl cyclase as well as G protein immunoreactivity in the myocardium from young and senescent F-344 rats with and without prior exercise training by treadmill running. Isoproterenol, beta-gamma-imidoguanosine 5'-triphosphate, and forskolin stimulation of adenylyl cyclase activity declined by approximately 50% with age. Training increased the stimulation in the senescent rats and decreased the stimulation in the young rats such that the age-related decline in signal transduction was no longer significant. Gs alpha protein immunoreactivity was unchanged by age or training. These data suggest that in young rats exercise training decreases beta-adrenergic signal transduction, whereas in older rats training increases signal transduction, partially offsetting the decline in signal transduction with age.

Adenylyl Cyclases↗
PubMed1994

Forskolin binding sites and G-protein immunoreactivity in rat hearts during aging.

Although the number of beta-adrenoceptors is unchanged with age in rat heart, both beta-adrenoceptor and postreceptor activation of adenylyl cyclase decreases with age. Pharmacologic data suggest that it is the amount of adenylyl cyclase enzyme units that limit activation of adenylyl cyclase with senescence, but direct quantitation of either G protein or adenylyl cyclase in rat heart with age is lacking. To quantitate the amount of adenylyl cyclase and G proteins with age directly, we assessed forskolin-stimulated adenylyl cyclase activity, the number of [3H] forskolin binding sites, and stimulatory G protein (Gs alpha) and inhibitory G protein (Gi alpha) immunoreactivity in the ventricles from 6- and 24-month-old F-344 rats. The amount of Gs alpha and Gi alpha was unchanged with age in both crude membranes and partially purified membranes from ventricles. In contrast, there was a 32% decrease in the ability of forskolin to stimulate adenylyl cyclase maximally and a 41% decrease in the number of forskolin binding sites with age. Sensitivity for forskolin activation was unchanged with age, but there was a slight increase in affinity for [3H]forskolin binding. The decrease in the amount of adenylyl cyclase with age correlates with the diminished capacity to activate adenylyl cyclase with age and may account for the reduced beta-adrenergic signal transduction observed in senescent rat heart.

Adenylyl Cyclases↗
PubMed1993

Loss of mitochondrial hsp60 function: nonequivalent effects on matrix-targeted and intermembrane-targeted proteins.

We have created yeast strains in which the mitochondrial chaperonin, hsp60, can be either physically depleted or functionally inactivated. Cells completely depleted of hsp60 stop growing but retain for awhile the capacity to reaccumulate hsp60. While this newly made hsp60 is targeted to and processed correctly within the mitochondrion, assembly of a functional hsp60 complex does not occur. Rather, the hsp60 monomers are localized in different-size soluble complexes containing another mitochondrial chaperone, the mitochondrial form of hsp70. A number of other mitochondrial matrix-targeted proteins synthesized in the absence of functional hsp60 are imported into mitochondria but often show some buildup of precursor forms and, unlike hsp60, accumulate as insoluble aggregates. By contrast, several mitochondrial proteins normally targeted to the intermembrane space show normal processing in the complete absence of a functional hsp60 complex. Similar and complementary results were obtained when we examined the metabolism of matrix- and intermembrane space-localized proteins in cells expressing three different temperature-sensitive alleles of HSP60. In all cases, matrix-targeted proteins synthesized at nonpermissive (i.e., hsp60-inactivating) temperatures were correctly targeted to and processed within mitochondria but accumulated predominantly or totally as insoluble aggregates. The metabolism of two intermembrane space proteins, cytochrome b2 and cytochrome c1, was unaffected at the nonpermissive temperature, as judged by the correct processing and complete solubility of newly synthesized forms of both proteins. These findings are discussed with regard to current models of intermembrane targeting.

Blotting, Western↗
PubMed1989

Chronotropic effect of the methanolic extracts of the plants of the Paris species and steroidal glycosides isolated from P. vietnamensis on spontaneous beating of myocardial cells.

Rhizomes of five identified plants of the Paris species, Liliaceae, and Rhizoma Paridis which are sold as a crude drug named "Zao Xiu," "Qiyeyizhihua" or other names in nine different markets in China were tested for their effects on cultured cardiomyocytes. In the standard medium, eight methanol extracts out of sixteen at a concentration of 0.2 mg/ml stopped the spontaneous beating of myocardial cell sheets, but these extracts significantly increased the beating rate when the concentration was reduced to one half. In the culture medium with a low calcium concentration, 0.5 mM, the beating rate of the cells decreased to about 60% of that of the control in the standard medium. The addition of five of the extracts to the low calcium medium at a concentration of 0.1 mg/ml caused a stop of cell beating, but the other extracts increased beating rate at least by 10%. These steroidal glycosides isolated from the rhyzomes of P. vietnamensis (Takht.) H. Li also stimulated cell beating. Among them, diosgenin-3)-alpha-L-rhamno-pyranosyl-(1---2)-(alpha-L-arabinofura nosyl-(1---4))-D-glucopranoside (compound 1) was the most effective stimulant for cell beating as well as calcium uptake by the myocardial cells.

Animals↗