scholarly journals The WD-repeats of Net2p Interact with Dnm1p and Fis1p to Regulate Division of Mitochondria

2003 ◽  
Vol 14 (10) ◽  
pp. 4126-4139 ◽  
Author(s):  
Kara L. Cerveny ◽  
Robert E. Jensen
Keyword(s):  
Mitochondrial Fission ◽  
Terminal Region ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Dominant Negative Effect ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Wd Repeats

The Net2, Fis1, and Dnm1 proteins are required for the division of mitochondria in the yeast Saccharomyces cerevisiae. Net2p has an amino-terminal region that contains predicted coiled-coil motifs and a carboxyl-terminal domain composed of WD-40 repeats. We found that the amino-terminal part of Net2p interacts with Fis1p, whereas the carboxyl-terminal region interacts with both Dnm1p and Fis1p. Overproduction of either domain of Net2p in yeast cells poisons mitochondrial fission, and the dominant-negative effect caused by the WD-repeats of Net2p is suppressed by increased levels of Dnm1p. Point mutations in the WD-region of Net2p or in the GTPase region of Dnm1p disrupt the normal Net2p-Dnm1p interaction, causing Net2p to lose its normal punctate distribution. Our results suggest that Dnm1p interacts with the WD-repeats of Net2p and in a GTP-dependent manner recruits Net2p to sites of mitochondrial division. Furthermore, our results indicate that Net2p is required for proper assembly of the mitochondrial fission components to regulate organelle division.

scholarly journals SUMO chain formation relies on the amino-terminal region of SUMO-conjugating enzyme and has dedicated substrates in plants

2018 ◽  
Vol 475 (1) ◽  
pp. 61-74 ◽  
Author(s):  
Konstantin Tomanov ◽  
Lilian Nehlin ◽  
Ionida Ziba ◽  
Andreas Bachmair
Keyword(s):  
Terminal Region ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
In Vitro Activity ◽  
Chain Formation ◽  
Amino Terminal ◽  
Sumo Conjugation ◽  
Negative Effect ◽  
Conjugating Enzyme

The small ubiquitin-related modifier (SUMO) conjugation apparatus usually attaches single SUMO moieties to its substrates, but SUMO chains have also been identified. To better define the biochemical requirements and characteristics of SUMO chain formation, mutations in surface-exposed Lys residues of Arabidopsis SUMO-conjugating enzyme (SCE) were tested for in vitro activity. Lys-to-Arg changes in the amino-terminal region of SCE allowed SUMO acceptance from SUMO-activating enzyme and supported substrate mono-sumoylation, but these mutations had significant effects on SUMO chain assembly. We found no indication that SUMO modification of SCE promotes chain formation. A substrate was identified that is modified by SUMO chain addition, showing that SCE can distinguish substrates for either mono-sumoylation or SUMO chain attachment. It is also shown that SCE with active site Cys mutated to Ser can accept SUMO to form an oxyester, but cannot transfer this SUMO moiety onto substrates, explaining a previously known dominant negative effect of this mutation.

Effects of truncated neurofilament proteins on the endogenous intermediate filaments in transfected fibroblasts

1991 ◽  
Vol 99 (2) ◽  
pp. 335-350 ◽  
Author(s):  
S.S. Chin ◽  
P. Macioce ◽  
R.K. Liem
Keyword(s):  
Intermediate Filament ◽  
Dominant Negative ◽  
Deletion Mutants ◽  
Tail Domain ◽  
Cultured Fibroblasts ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Novel Approach ◽  
Carboxyl Terminal

The expression and assembly characteristics of carboxyl- and amino-terminal deletion mutants of rat neurofilament low Mr (NF-L) and neurofilament middle Mr (NF-M) proteins were examined by transient transfection of cultured fibroblasts. Deletion of the carboxyl-terminal tail domain of either protein indicated that this region was not absolutely essential for co-assembly into the endogenous vimentin cytoskeleton. However, deletion into the alpha-helical rod domain resulted in an inability of the mutant proteins to co-assemble with vimentin into filamentous structures. Instead, the mutant proteins appeared to be assembled into unusual tubular-vesicular structures. Additionally, these latter deletions appeared to act as dominant negative mutants which induced the collapse of the endogenous vimentin cytoskeleton as well as the constitutively expressed NF-H and NF-M cytoskeletons in stably transfected cell lines. Thus, an intact alpha-helical rod domain was essential for normal IF co-assembly whereas carboxyl-terminal deletions into this region resulted in dramatic alterations of the existing type III and IV intermediate filament cytoskeletons in vivo. Deletions from the amino-terminal end into the alpha-helical rod region gave different results. With these deletions, the transfected protein was not co-assembled into filaments and the endogenous vimentin IF network was not disrupted, indicating that these deletion mutants are recessive. The dominant negative mutants may provide a novel approach to studying intermediate filament function within living cells.

Methylenetetrahydrofolate dehydrogenase – methenyltetrahydrofolate cyclohydrolase – formyltetrahydrofolate synthetase from porcine liver Location of the activities in two domains of the multifunctional polypeptide

1979 ◽  
Vol 57 (6) ◽  
pp. 806-812 ◽  
Author(s):  
L. U. L. Tan ◽  
R. E. MacKenzie
Keyword(s):  
Terminal Region ◽  
Synthetase Activity ◽  
Terminal Portion ◽  
Porcine Liver ◽  
Pig Liver ◽  
Formyltetrahydrofolate Synthetase ◽  
Partial Identity ◽  
Amino Terminal ◽  
Methylenetetrahydrofolate Dehydrogenase ◽  
Carboxyl Terminal

Chymotryptic cleavage of the trifunctional protein methylenetetrahydrofolate dehydrogenase – methenyltetrahydrofolate cyclohydrolase – formyltetrahydrofolate synthetase from pig liver yields a fragment of two-thirds the original polypeptide that retains only synthetase activity. A smaller polypeptide corresponding to about one-third of the original polypeptide was shown earlier to retain dehydrogenase–cyclohydrolase activity. On immunodiffusion, the synthetase fragment cross-reacts and shows partial identity with antibodies raised against the uncleaved enzyme but shows nonidentity with the dehydrogenase–cyclohydrolase fragment, suggesting that the two fragments are derived from different regions of the polypeptide. Amino-terminal analysis of the peptides and uncleaved enzyme indicate that the dehydrogenase–cyclohydrolase activities are located at the amino-terminal region and the synthetase near the carboxyl-terminal portion of the polypeptide.

scholarly journals The multiple RNA-binding domains of the mRNA poly(A)-binding protein have different RNA-binding activities.

1991 ◽  
Vol 11 (7) ◽  
pp. 3419-3424 ◽  
Author(s):  
C G Burd ◽  
E L Matunis ◽  
G Dreyfuss
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Binding Activity ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal ◽  
Binding Domains ◽  
Rna Binding Domains

The poly(A)-binding protein (PABP) is the major mRNA-binding protein in eukaryotes, and it is essential for viability of the yeast Saccharomyces cerevisiae. The amino acid sequence of the protein indicates that it consists of four ribonucleoprotein consensus sequence-containing RNA-binding domains (RBDs I, II, III, and IV) and a proline-rich auxiliary domain at the carboxyl terminus. We produced different parts of the S. cerevisiae PABP and studied their binding to poly(A) and other ribohomopolymers in vitro. We found that none of the individual RBDs of the protein bind poly(A) specifically or efficiently. Contiguous two-domain combinations were required for efficient RNA binding, and each pairwise combination (I/II, II/III, and III/IV) had a distinct RNA-binding activity. Specific poly(A)-binding activity was found only in the two amino-terminal RBDs (I/II) which, interestingly, are dispensable for viability of yeast cells, whereas the activity that is sufficient to rescue lethality of a PABP-deleted strain is in the carboxyl-terminal RBDs (III/IV). We conclude that the PABP is a multifunctional RNA-binding protein that has at least two distinct and separable activities: RBDs I/II, which most likely function in binding the PABP to mRNA through the poly(A) tail, and RBDs III/IV, which may function through binding either to a different part of the same mRNA molecule or to other RNA(s).

Application of a sequence-specific radioimmunoassay for the carboxyl-terminal region of corticotropin.

1981 ◽  
Vol 27 (4) ◽  
pp. 549-552 ◽  
Author(s):  
L H Lazarus ◽  
R P DiAugustine ◽  
M N Khan ◽  
G D Jahnke ◽  
M D Erisman
Keyword(s):  
Molecular Mass ◽  
Human Plasma ◽  
Silicic Acid ◽  
Peptide Bond ◽  
Terminal Region ◽  
Carboxyl Terminus ◽  
Proteolytic Degradation ◽  
Amino Terminal ◽  
Specific Radioimmunoassay ◽  
Carboxyl Terminal

Abstract The carboxyl terminal region of corticotropin (ACTH), (Ala34-Phe-Pro-Glu-Leu-Phe39), a region of the hormone conserved during evolution, served as an antigen for the production of a sequence-specific antiserum. In a radioimmunoassay, peptides that extend toward the amino terminal from Ala34, such as [Tyr,Gly]-ACTH34-39, ACTH18-39, and ACTH, had greater affinity for the antibody, which suggests that the antiserum recognizes the peptide bond preceding the alanyl residue. The assay readily detects 30 to 50 pmol of ACTH per liter with an incubation of only 3 h, and the antiserum cross reacts with larger molecular mass forms of the hormone. The amount of immunoreactive ACTH extracted by adsorption onto silicic acid from rat and human plasma was only 0.36 to 0.79 of that detected by a mid-region ACTH assay, which suggests proteolytic degradation at the carboxyl terminus of ACTH.

scholarly journals Recycling of proteins from the Golgi compartment to the ER in yeast.

1990 ◽  
Vol 111 (2) ◽  
pp. 369-377 ◽  
Author(s):  
N Dean ◽  
H R Pelham
Keyword(s):  
Fusion Protein ◽  
Recognition System ◽  
Dependent Manner ◽  
Terminal Sequence ◽  
Yeast Saccharomyces Cerevisiae ◽  
Subcellular Organelles ◽  
Er Retention ◽  
Golgi Compartment ◽  
Carboxyl Terminal

In the yeast Saccharomyces cerevisiae, the carboxyl terminal sequence His-Asp-Glu-Leu (HDEL) has been shown to function as an ER retention sequence (Pelham, H. R. B., K. G. Hardwick, and M. J. Lewis. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1757-1762). To examine the mechanism of retention of soluble ER proteins in yeast, we have analyzed the expression of a preproalpha factor fusion protein, tagged at the carboxyl terminus with the HDEL sequence. We demonstrate that this fusion protein, expressed in vivo, accumulates intracellularly as a precursor containing both ER and Golgi-specific oligosaccharide modifications. The Golgi-specific carbohydrate modification, which occurs in a SEC18-dependent manner, consists of alpha 1-6 mannose linkages, with no detectable alpha 1-3 mannose additions, indicating that the transit of the HDEL-tagged fusion protein is confined to an early Golgi compartment. Results obtained from the fractionation of subcellular organelles from yeast expressing HDEL-tagged fusion proteins suggest that the Golgi-modified species are present in the ER. Overexpression of HDEL-tagged preproalpha factor results in the secretion of an endogenous HDEL-containing protein, demonstrating that the HDEL recognition system can be saturated. These results support the model in which the retention of these proteins in the ER is dependent on their receptor-mediated recycling from the Golgi complex back to the ER.

scholarly journals Identification of Epitope and Surface-Exposed Domains of Shigella flexneri Invasion Plasmid Antigen D (IpaD)

1998 ◽  
Vol 66 (5) ◽  
pp. 1999-2006 ◽  
Author(s):  
K. Ross Turbyfill ◽  
Jennifer A. Mertz ◽  
Corey P. Mallett ◽  
Edwin V. Oaks
Keyword(s):  
Amino Acid ◽  
Shigella Flexneri ◽  
Serum Antibody ◽  
Terminal Region ◽  
Virulence Plasmid ◽  
Amino Acid Residues ◽  
Unique Region ◽  
Amino Terminal ◽  
Convalescent Phase ◽  
Carboxyl Terminal

ABSTRACT Transport and surface expression of the invasion plasmid antigens (Ipa proteins) is an essential trait in the pathogenicity ofShigella spp. In addition to the type III protein secretion system encoded by the mxi/spa loci on the large virulence plasmid, transport of IpaB and IpaC into the surrounding medium is modulated by IpaD. To characterize the structural topography of IpaD, the Geysen epitope-mapping system was used to identify epitopes recognized by surface-reactive monoclonal and polyclonal antibodies produced against purified recombinant IpaD or synthetic IpaD peptides. Surface-exposed epitopes of IpaD were confined to the first 180 amino acid residues, whereas epitopes in the carboxyl-terminal half were not exposed on the Shigella surface. By using convalescent-phase sera from 10 Shigella flexneri-infected monkeys, numerous epitopes were mapped within a surface-exposed region of IpaD between amino acid residues 14 and 77. Epitopes were also identified in the carboxyl-terminal half of IpaD with a few convalescent-phase sera. Comparison of IpaD epitope sequences withSalmonella SipD sequences indicated that very similar epitopes may exist in the carboxyl-terminal region of each protein whereas the IpaD epitopes in the surface-exposed amino-terminal region were unique for the Shigella protein. Although the IpaD and SipD homologs may play similar roles in transport, the dominant serum antibody response to IpaD is against the unique region of this protein exposed on the surface of the pathogen.

scholarly journals Size of Cells and Physicochemical Properties of Membranes are Related to Flavor Production during Sake Brewing in the Yeast Saccharomyces cerevisiae

Membranes ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 440
Author(s):  
Tsuyoshi Yoda ◽  
Tomoaki Saito
Keyword(s):  
Isoamyl Alcohol ◽  
Lipid Vesicles ◽  
Caproic Acid ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Concentration Dependent Manner ◽  
Generalized Polarization ◽  
Flavor Components ◽  
The Impact

Ethyl caproate (EC) and isoamyl acetate (IA) are key flavor components of sake. Recently, attempts have been made to increase the content of good flavor components, such as EC and IA, in sake brewing. However, the functions of EC and IA in yeast cells remain poorly understood. Therefore, we investigated the effects of EC and IA using cell-sized lipid vesicles. We also investigated lipid vesicles containing EC and/or caproic acid (CA) as well as IA and/or isoamyl alcohol (IAA). CA and IAA are precursors of EC and IA, respectively, and are important flavors in sake brewing. The size of a vesicle is influenced by flavor compounds and their precursors in a concentration-dependent manner. We aimed to establish the conditions in which the vesicles contained more flavors simultaneously and with different ratios. Interestingly, vesicles were largest in a mixture of 50% of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with 25% EC and 25% CA or a mixture of 50% DOPC with 25% IA and 25% IAA. The impact of flavor additives on membrane fluidity was also studied using Laurdan generalized polarization. During the production process, flavors may regulate the fluidity of lipid membranes.

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