scholarly journals Structural and functional characterization of Sec66p, a new subunit of the polypeptide translocation apparatus in the yeast endoplasmic reticulum.

1993 ◽  
Vol 4 (9) ◽  
pp. 931-939 ◽  
Author(s):  
D Feldheim ◽  
K Yoshimura ◽  
A Admon ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Functional Characterization ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Endoplasmic Reticulum Membrane ◽  
Restrictive Temperature ◽  
Permissive Temperature

SEC66 encodes the 31.5-kDa glycoprotein of the Sec63p complex, an integral endoplasmic reticulum membrane protein complex required for translocation of presecretory proteins in Saccharomyces cerevisiae. DNA sequence analysis of SEC66 predicts a 23-kDa protein with no obvious NH2-terminal signal sequence but with one domain of sufficient length and hydrophobicity to span a lipid bilayer. Antibodies directed against a recombinant form of Sec66p were used to confirm the membrane location of Sec66p and that Sec66p is a glycoprotein of 31.5 kDa. A null mutation in SEC66 renders yeast cells temperature sensitive for growth. sec66 cells accumulate some secretory precursors at a permissive temperature and a variety of precursors at the restrictive temperature. sec66 cells show defects in Sec63p complex formation. Because sec66 cells affect the translocation of some, but not all secretory precursor polypeptides, the role of Sec66p may be to interact with the signal peptide of presecretory proteins.

scholarly journals An ATP-binding membrane protein is required for protein translocation across the endoplasmic reticulum membrane.

1991 ◽  
Vol 2 (10) ◽  
pp. 851-859 ◽  
Author(s):  
D L Zimmerman ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Microsomal Membrane ◽  
Atp Binding ◽  
Endoplasmic Reticulum Membrane ◽  
Receptor Complex

The role of nucleotides in providing energy for polypeptide transfer across the endoplasmic reticulum (ER) membrane is still unknown. To address this question, we treated ER-derived mammalian microsomal vesicles with a photoactivatable analogue of ATP, 8-N3ATP. This treatment resulted in a progressive inhibition of translocation activity. Approximately 20 microsomal membrane proteins were labeled by [alpha 32P]8-N3ATP. Two of these were identified as proteins with putative roles in translocation, alpha signal sequence receptor (SSR), the 35-kDa subunit of the signal sequence receptor complex, and ER-p180, a putative ribosome receptor. We found that there was a positive correlation between inactivation of translocation activity and photolabeling of alpha SSR. In contrast, our data demonstrate that the ATP-binding domain of ER-p180 is dispensable for translocation activity and does not contribute to the observed 8-N3ATP sensitivity of the microsomal vesicles.

Biochemical differentiation in a mutant of Dictyostelium discoideum defective in cyclic AMP chemotaxis and in intercellular cohesion

Development ◽  
1989 ◽  
Vol 107 (1) ◽  
pp. 153-163
Author(s):  
U.K. Srinivas ◽  
E.J. Henderson
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Regulation Of Gene Expression ◽  
Temperature Sensitive ◽  
Morphological Development ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Beta Glucosidase ◽  
Biochemical Differentiation

A temperature-sensitive mutant of Dictyostelium discoideum has been isolated based on its lack of chemotaxis toward cyclic AMP at the restrictive temperature, 27 degrees C. The mutant develops normally at the permissive temperature, 22 degrees C, but fails to aggregate or complete development at the restrictive temperature. The temperature-sensitive phenotype can be bypassed by allowing cultures to grown into late log phase or to starve for 60–90 min at 22 degrees C prior to a shift to 27 degrees C. At 27 degrees C, the mutant overproduces cell surface cyclic AMP receptors of both high and low affinity and is capable of spontaneous oscillations in light scattering in cell suspensions. Despite its complete lack of morphological development, the mutant undergoes extensive biochemical differentiation. At the onset of starvation, it shows increased levels of N-acetylglucosaminidase, it express cyclic AMP receptors at the normal time and, although somewhat slowly, suppresses those receptors as if aggregation had been achieved. Metabolic pulse labellings with [35S]methionine revealed that the mutant at 27 degrees C displays the same changes in the patterns of newly synthesized proteins observed during the vegetative-to-aggregation and the aggregation-to-slug stages of normal development. The only clear difference from wild type was the failure of the culmination-stage isozyme of beta-glucosidase to appear. The mutant is defective in establishment of intercellular cohesion mechanisms, correlated with poor agglutination by concanavalin A, at the restrictive temperature. The properties of the mutant place severe constraints on models regarding the role of chemoreception and intercellular cohesion in regulation of gene expression.

Functional characterization of soybean (Glycine max) DIRIGENT genes reveals an important role of GmDIR27 in the regulation of pod dehiscence

Genomics ◽  
2020 ◽  
Author(s):  
Xiaofei Ma ◽  
Wenying Xu ◽  
Tong Liu ◽  
Ruying Chen ◽  
Hong Zhu ◽  
...  
Keyword(s):  
Glycine Max ◽  
Functional Characterization ◽  
Pod Dehiscence

scholarly journals Molecular and Functional Characterization of a ToxR-Regulated Lipoprotein from a Clinical Isolate of Aeromonas hydrophila

2006 ◽  
Vol 74 (7) ◽  
pp. 3742-3755 ◽  
Author(s):  
Lakshmi Pillai ◽  
Jian Sha ◽  
Tatiana E. Erova ◽  
Amin A. Fadl ◽  
Bijay K. Khajanchi ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Aeromonas Hydrophila ◽  
Functional Characterization ◽  
Affinity Column ◽  
Serum Resistance ◽  
Classical Pathway ◽  
Dependent Manner ◽  
T7 Promoter

ABSTRACT Human diseases caused by species of Aeromonas have been classified into two major groups: septicemia and gastroenteritis. In this study, we reported the molecular and functional characterization of a new virulence factor, ToxR-regulated lipoprotein, or TagA, from a diarrheal isolate, SSU, of Aeromonas hydrophila. The tagA gene of A. hydrophila exhibited 60% identity with that of a recently identified stcE gene from Escherichia coli O157:H7, which encoded a protein (StcE) that provided serum resistance to the bacterium and prevented erythrocyte lysis by controlling classical pathway of complement activation by cleaving the complement C1-esterase inhibitor (C1-INH). We purified A. hydrophila TagA as a histidine-tagged fusion protein (rTagA) from E. coli DE3 strain using a T7 promoter-based pET30 expression vector and nickel affinity column chromatography. rTagA cleaved C1-INH in a time-dependent manner. The tagA isogenic mutant of A. hydrophila, unlike its corresponding wild-type (WT) or the complemented strain, was unable to cleave C1-INH, which is required to potentiate the C1-INH-mediated lysis of host and bacterial cells. We indeed demonstrated colocalization of C1-INH and TagA on the bacterial surface by confocal fluorescence microscopy, which ultimately resulted in increased serum resistance of the WT bacterium. Likewise, we delineated the role of TagA in contributing to the enhanced ability of C1-INH to inhibit the classical complement-mediated lysis of erythrocytes. Importantly, we provided evidence that the tagA mutant was significantly less virulent in a mouse model of infection (60%) than the WT bacterium at two 50% lethal doses, which resulted in 100% mortality within 48 h. Taken together, our data provided new information on the role of TagA as a virulence factor in bacterial pathogenesis. This is the first report of TagA characterization from any species of Aeromonas.

scholarly journals Functional Characterization of Spliceosomal Introns and Identification of U2, U4, and U5 snRNAs in the Deep-Branching Eukaryote Entamoeba histolytica

2007 ◽  
Vol 6 (6) ◽  
pp. 940-948 ◽  
Author(s):  
Carrie A. Davis ◽  
Michael P. S. Brown ◽  
Upinder Singh
Keyword(s):  
Entamoeba Histolytica ◽  
Splice Site ◽  
Expressed Sequence Tag ◽  
Functional Characterization ◽  
Molecular Evidence ◽  
Spliceosomal Introns ◽  
Accurate Expression ◽  
Eukaryotic Organisms

ABSTRACT Pre-mRNA splicing is essential to ensure accurate expression of many genes in eukaryotic organisms. In Entamoeba histolytica, a deep-branching eukaryote, approximately 30% of the annotated genes are predicted to contain introns; however, the accuracy of these predictions has not been tested. In this study, we mined an expressed sequence tag (EST) library representing 7% of amoebic genes and found evidence supporting splicing of 60% of the testable intron predictions, the majority of which contain a GUUUGU 5′ splice site and a UAG 3′ splice site. Additionally, we identified several splice site misannotations, evidence for the existence of 30 novel introns in previously annotated genes, and identified novel genes through uncovering their spliced ESTs. Finally, we provided molecular evidence for the E. histolytica U2, U4, and U5 snRNAs. These data lay the foundation for further dissection of the role of RNA processing in E. histolytica gene expression.

scholarly journals Cloning and functional characterization of mammalian homologues of the COPII component Sec23.

1996 ◽  
Vol 7 (10) ◽  
pp. 1535-1546 ◽  
Author(s):  
J P Paccaud ◽  
W Reith ◽  
J L Carpentier ◽  
M Ravazzola ◽  
M Amherdt ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Cdna Clones ◽  
Restrictive Temperature ◽  
Rnase Protection ◽  
Cell Extracts ◽  
Exact Function ◽  
Mammalian Protein

We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa. hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p. Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p. Cytosolic hSec23Ap migrated with an apparent molecular weight of 350 kDa on a gel filtration column, suggesting that it is part of a protein complex. By immunoelectron microscopy, hSec23Ap was found essentially in the ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles. hSec23Ap is a functional homologue of the yeast Sec23p as the hSec23A isoform complemented the temperature sensitivity of the Saccharomyces cerevisiae sec23-1 mutation at a restrictive temperature of 34 degrees C. RNase protection assays indicated that both hSec23 isoforms are coexpressed in various human tissues, although at a variable ratio. Our data demonstrate that hSec23Ap is the functional human counterpart of the yeast COPII component Sec23p and suggest that it plays a similar role in mammalian protein export from the ER. The exact function of hSec23Bp remains to be determined.

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