scholarly journals Participation of the proteasomal lid subunit Rpn11 in mitochondrial morphology and function is mapped to a distinct C-terminal domain

2004 ◽  
Vol 381 (1) ◽  
pp. 275-285 ◽  
Author(s):  
Teresa RINALDI ◽  
Elah PICK ◽  
Alessia GAMBADORO ◽  
Stefania ZILLI ◽  
Vered MAYTAL-KIVITY ◽  
...  
Keyword(s):  
Terminal Region ◽  
Proper Function ◽  
Mitochondrial Morphology ◽  
Wild Type ◽  
Polyubiquitin Chains ◽  
Unique Role ◽  
Terminal Domain ◽  
C Terminus ◽  
Tubular System ◽  
And Function

Substrates destined for degradation by the 26 S proteasome are labelled with polyubiquitin chains. Rpn11/Mpr1, situated in the lid subcomplex, partakes in the processing of these chains or in their removal from substrates bound to the proteasome. Rpn11 also plays a role in maintaining mitochondrial integrity, tubular structure and proper function. The recent finding that Rpn11 participates in proteasome-associated deubiquitination focuses interest on the MPN+ (Mpr1, Pad1, N-terminal)/JAMM (JAB1/MPN/Mov34) metalloprotease site in its N-terminal domain. However, Rpn11 damaged at its C-terminus (the mpr1-1 mutant) causes pleiotropic effects, including proteasome instability and mitochondrial morphology defects, resulting in both proteolysis and respiratory malfunctions. We find that overexpression of WT (wild-type) RPN8, encoding a paralogous subunit that does not contain the catalytic MPN+ motif, corrects proteasome conformations and rescues cell cycle phenotypes, but is unable to correct defects in the mitochondrial tubular system or respiratory malfunctions associated with the mpr1-1 mutation. Transforming mpr1-1 with various RPN8–RPN11 chimaeras or with other rpn11 mutants reveals that a WT C-terminal region of Rpn11 is necessary, and more surprisingly sufficient, to rescue the mpr1-1 mitochondrial phenotype. Interestingly, single-site mutants in the catalytic MPN+ motif at the N-terminus of Rpn11 lead to reduced proteasome-dependent deubiquitination connected with proteolysis defects. Nevertheless, these rpn11 mutants suppress the mitochondrial phenotypes associated with mpr1-1 by intragene complementation. Together, these results point to a unique role for the C-terminal region of Rpn11 in mitochondrial maintenance that may be independent of its role in proteasome-associated deubiquitination.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

scholarly journals C-terminal region regulates the functional expression of human noradrenaline transporter splice variants

2006 ◽  
Vol 401 (1) ◽  
pp. 185-195 ◽  
Author(s):  
Chiharu Sogawa ◽  
Kei Kumagai ◽  
Norio Sogawa ◽  
Katsuya Morita ◽  
Toshihiro Dohi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Splice Variants ◽  
Functional Expression ◽  
Surface Expression ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
C Terminus ◽  
And Function

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl−-dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

scholarly journals The N-Terminus ofDictyosteliumScar Interacts with Abi and HSPC300 and Is Essential for Proper Regulation and Function

2007 ◽  
Vol 18 (5) ◽  
pp. 1609-1620 ◽  
Author(s):  
Diana Caracino ◽  
Cheryl Jones ◽  
Mark Compton ◽  
Charles L. Saxe
Keyword(s):  
Actin Polymerization ◽  
Terminal Region ◽  
Wild Type ◽  
Large Molecular Weight ◽  
Weight Protein ◽  
And Function ◽  
Necessary And Sufficient

Scar/WAVE proteins, members of the conserved Wiskott-Aldrich syndrome (WAS) family, promote actin polymerization by activating the Arp2/3 complex. A number of proteins, including a complex containing Nap1, PIR121, Abi1/2, and HSPC300, interact with Scar/WAVE, though the role of this complex in regulating Scar function remains unclear. Here we identify a short N-terminal region of Dictyostelium Scar that is necessary and sufficient for interaction with HSPC300 and Abi in vitro. Cells expressing Scar lacking this N-terminal region show abnormalities in F-actin distribution, cell morphology, movement, and cytokinesis. This is true even in the presence of wild-type Scar. The data suggest that the first 96 amino acids of Scar are necessary for participation in a large-molecular-weight protein complex, and that this Scar-containing complex is responsible for the proper localization and regulation of Scar. The presence of mis-regulated or unregulated Scar has significant deleterious effects on cells and may explain the need to keep Scar activity tightly controlled in vivo either by assembly in a complex or by rapid degradation.

scholarly journals Human aldolase A natural mutants: relationship between flexibility of the C-terminal region and enzyme function

2004 ◽  
Vol 380 (1) ◽  
pp. 51-56 ◽  
Author(s):  
Gabriella ESPOSITO ◽  
Luigi VITAGLIANO ◽  
Paola COSTANZO ◽  
Loredana BORRELLI ◽  
Rita BARONE ◽  
...  
Keyword(s):  
Terminal Region ◽  
Arthrogryposis Multiplex Congenita ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Subunit Interface ◽  
Graphic Analysis ◽  
Fructose 1,6 Bisphosphate ◽  
And Function ◽  
Functional Profiles ◽  
Aldolase A

We have identified a new mutation in the FBP (fructose 1,6-bisphosphate) aldolase A gene in a child with suspected haemolytic anaemia associated with myopathic symptoms at birth and with a subsequent diagnosis of arthrogryposis multiplex congenita and pituitary ectopia. Sequence analysis of the whole gene, also performed on the patient's full-length cDNA, revealed only a Gly346→Ser substitution in the heterozygous state. We expressed in a bacterial system the new aldolase A Gly346→Ser mutant, and the Glu206→Lys mutant identified by others, in a patient with an aldolase A deficit. Analysis of their functional profiles showed that the Gly346→Ser mutant had the same Km as the wild-type enzyme, but a 4-fold lower kcat. The Glu206→Lys mutant had a Km approx. 2-fold higher than that of both the Gly346→Ser mutant and the wild-type enzyme, and a kcat value 40% less than the wild-type. The Gly346→Ser and wild-type enzymes had the same Tm (melting temperature), which was approx. 6–7 °C higher than that of the Glu206→Lys enzyme. An extensive molecular graphic analysis of the mutated enzymes, using human and rabbit aldolase A crystallographic structures, suggests that the Glu206→Lys mutation destabilizes the aldolase A tetramer at the subunit interface, and highlights the fact that the glycine-to-serine substitution at position 346 limits the flexibility of the C-terminal region. These results also provide the first evidence that Gly346 is crucial for the correct conformation and function of aldolase A, because it governs the entry/release of the substrates into/from the enzyme cleft, and/or allows important C-terminal residues to approach the active site.

scholarly journals The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions

2016 ◽  
Vol 198 (16) ◽  
pp. 2263-2274 ◽  
Author(s):  
Ryan N. C. Buensuceso ◽  
Ylan Nguyen ◽  
Kun Zhang ◽  
Martin Daniel-Ivad ◽  
Seiji N. Sugiman-Marangos ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cyclic Amp ◽  
Tetratricopeptide Repeat ◽  
Twitching Motility ◽  
Wild Type ◽  
Inner Membrane ◽  
Content Type ◽  
Terminal Domain ◽  
C Terminus ◽  
Independent Functions

ABSTRACTFimV is aPseudomonas aeruginosainner membrane protein that regulates intracellular cyclic AMP (cAMP) levels—and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)—by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the “FimV C-terminal domain,” which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861–919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689failed to restore wild-type motility in afimVmutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions.IMPORTANCEFimV is important for twitching motility and cAMP-dependent virulence gene expression inP. aeruginosa. FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.

scholarly journals Role of the N- and C-terminal regions of FliF, the MS ring component in Vibrio flagellar basal body

2021 ◽  
Author(s):  
Seiji Kojima ◽  
Hiroki Kajino ◽  
Keiichi Hirano ◽  
Yuna Inoue ◽  
Hiroyuki Terashima ◽  
...  
Keyword(s):  
Basal Body ◽  
Ring Formation ◽  
Terminal Region ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Bacterial Flagellum ◽  
Transmembrane Segments ◽  
Ring Assembly ◽  
C Terminus ◽  
N Terminus

AbstractThe MS ring is a part of the flagellar basal body and formed by 34 subunits of FliF, which consists of a large periplasmic region and two transmembrane segments connected to the N- and C-terminal regions facing the cytoplasm. A cytoplasmic protein, FlhF, which determines the position and number of the basal body, supports MS ring formation in the membrane. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues at the N-terminus (ΔN30 and ΔN50), and 83 (ΔC83) or 110 residues (ΔC110) at the C-terminus. The N-terminal deletions were functional and conferred motility of Vibrio cells, whereas the C-terminal deletions were nonfunctional. The mutants were expressed in Escherichia coli to determine whether an MS ring could still be assembled. When co-expressing ΔN30FliF or ΔN50FliF with FlhF, fewer MS rings were observed than with the expression of wild-type FliF, in the MS ring fraction, suggesting that the N-terminus interacts with FlhF. MS ring formation is probably inefficient without an additional factor or FlhF. The deletion of the C-terminal cytoplasmic region did not affect the ability of FliF to form an MS ring because a similar number of MS rings were observed for ΔC83FliF as with wild-type FliF, although further deletion of the second transmembrane segment (ΔC110FliF) abolished it. These results suggest that the terminal regions of FliF have distinct roles; the N-terminal region for efficient MS ring formation and the C-terminal region for MS ring function. The second transmembrane segment is indispensable for MS ring assembly.ImportanceThe bacterial flagellum is a supramolecular architecture involved in cell motility. At the base of the flagella, a rotary motor that begins to construct an MS ring in the cytoplasmic membrane comprises 34 transmembrane proteins (FliF). Here, we investigated the roles of the N and C terminal regions of FliF, which are MS rings. Unexpectedly, the cytoplasmic regions of FliF are not indispensable for the formation of the MS ring, but the N-terminus appears to assist in ring formation through recruitment of FlhF, which is essential for flagellar formation. The C-terminus is essential for motor formation or function.

scholarly journals NSC348884 cytotoxicity is not mediated by inhibition of nucleophosmin oligomerization

2020 ◽  
Author(s):  
Markéta Šašinková ◽  
Petr Heřman ◽  
Aleš Holoubek ◽  
Dita Strachotová ◽  
Petra Otevřelová ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Cytotoxic Effect ◽  
Leukemia Cells ◽  
Live Cells ◽  
Wild Type ◽  
C Terminus ◽  
Interaction Partners ◽  
And Function ◽  
Acute Myeloid

AbstractOligomerization of the nucleolar phosphoprotein nucleophosmin (NPM) is mediated by its N-terminal domain. In acute myeloid leukemia, a frequent NPM mutation occurring at the C-terminus causes NPM delocalization to the cytoplasm. Due to formation of NPM heterooligomers, the wild-type NPM as well as many of NPM interaction partners are also delocalized. Proper localization and function of mislocalized proteins in the cells with mutated NPM may be restored by targeting NPM oligomerization. We introduce a reliable set of complementary methods for monitoring NPM oligomerization in both cell lysates and live cells. Using this methodological background we show that a putative inhibitor of NPM oligomerization, NSC348884, does not prevent formation of NPM oligomers in leukemia cells. Instead, we reveal that the observed cytotoxic effect of NSC348884 is associated with changes in cell adhesion signaling.

scholarly journals DROSOPHILA MTOR COMPLEX 2 PRESERVES MITOCHONDRIAL AND CARDIAC FUNCTION UNDER HIGH FAT DIET TREATMENT

2021 ◽  
Author(s):  
Kai Chang ◽  
Guillermo A. Requejo Figueroa ◽  
Hua Bai
Keyword(s):  
Mitochondrial Dysfunction ◽  
High Fat Diet ◽  
Mitochondrial Morphology ◽  
Contractile Dysfunction ◽  
Wild Type ◽  
High Fat ◽  
Mitochondrial Homeostasis ◽  
Mechanistic Target Of Rapamycin ◽  
And Function ◽  
Mtor Complex 1

AbstractHigh fat diet (HFD)-associated lipotoxicity is one of the major causes of cardiovascular diseases. The mechanistic target of rapamycin (mTOR) pathway, especially mTOR complex 1 (mTORC1), has been previously implicated in HFD-induced heart dysfunction. In the present study, we find that unlike mTORC1, mTOR complex 2 (mTORC2) protects hearts from HFD-induced cardiomyopathy and mitochondrial dysfunction in Drosophila. We show that HFD feeding induces contractile dysfunction along with altered mitochondrial morphology and function. Upon HFD feeding, the mitochondria of cardiomyocytes exhibit fragmentation, loss of membrane potential, and calcium overload. Interestingly, HFD feeding also reduces the activity of cardiac mTORC2. In line with this finding, the flies with cardiac-specific knockdown of rictor, the key subunit of mTORC2, show cardiac and mitochondrial dysfunction similar to what is observed in HFD-fed wild-type flies. Conversely, cardiac-specific activation of mTORC2 by overexpressing rictor attenuates HFD-induced mitochondrial and cardiac dysfunction. Thus, our findings suggest that mTORC2 is a cardioprotective factor and regulates mitochondrial homeostasis upon HFD feeding.

scholarly journals Differential Localization and Function of PB1-F2 Derived from Different Strains of Influenza A Virus

2010 ◽  
Vol 84 (19) ◽  
pp. 10051-10062 ◽  
Author(s):  
Chi-Jene Chen ◽  
Guang-Wu Chen ◽  
Ching-Ho Wang ◽  
Chih-Heng Huang ◽  
Yeau-Ching Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Amino Acid Sequences ◽  
Wild Type ◽  
Mitochondrial Localization ◽  
Sequence Context ◽  
C Terminus ◽  
Different Strains ◽  
And Function

ABSTRACT PB1-F2 is a viral protein that is encoded by the PB1 gene of influenza A virus by alternative translation. It varies in length and sequence context among different strains. The present study examines the functions of PB1-F2 proteins derived from various human and avian viruses. While H1N1 PB1-F2 was found to target mitochondria and enhance apoptosis, H5N1 PB1-F2, surprisingly, did not localize specifically to mitochondria and displayed no ability to enhance apoptosis. Introducing Leu into positions 69 (Q69L) and 75 (H75L) in the C terminus of H5N1 PB1-F2 drove 40.7% of the protein to localize to mitochondria compared with the level of mitochondrial localization of wild-type H5N1 PB1-F2, suggesting that a Leu-rich sequence in the C terminus is important for targeting of mitochondria. However, H5N1 PB1-F2 contributes to viral RNP activity, which is responsible for viral RNA replication. Lastly, although the swine-origin influenza virus (S-OIV) contained a truncated form of PB1-F2 (12 amino acids [aa]), potential mutation in the future may enable it to contain a full-length product. Therefore, the functions of this putative S-OIV PB1-F2 (87 aa) were also investigated. Although this PB1-F2 from the mutated S-OIV shares only 54% amino acid sequence identity with that of seasonal H1N1 virus, it also increased viral RNP activity. The plaque size and growth curve of the viruses with and without S-OIV PB1-F2 differed greatly. The PB1-F2 protein has various lengths, amino acid sequences, cellular localizations, and functions in different strains, which result in strain-specific pathogenicity. Such genetic and functional diversities make it flexible and adaptable in maintaining the optimal replication efficiency and virulence for various strains of influenza A virus.

scholarly journals Interaction between FliE and FlgB, a Proximal Rod Component of the Flagellar Basal Body ofSalmonella

2000 ◽  
Vol 182 (11) ◽  
pp. 3029-3036 ◽  
Author(s):  
Tohru Minamino ◽  
Shigeru Yamaguchi ◽  
Robert M. Macnab
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Basal Body ◽  
Strong Interactions ◽  
Wild Type ◽  
Body Protein ◽  
C Terminus ◽  
Genes Encoding ◽  
And Function ◽  
Better Than

ABSTRACT FliE is a flagellar basal body protein of Salmonellawhose detailed location and function have not been established. A mutant allele of fliE, which caused extremely poor flagellation and swarming, generated extragenic suppressors, all of which mapped to flgB, one of four genes encoding the basal body rod; the fliE flgB pseudorevertants were better flagellated and swarmed better than the fliE parent, especially when the temperature was reduced from 37 to 30°C. Motility of the pseudorevertants in liquid culture was markedly better than motility on swarm plates; we interpret this to mean that reduced flagellation is less deleterious at low viscous loads. Overproduction of the mutant FliE protein improved the motility of the parentalfliE mutant and its pseudorevertants, though not to wild-type levels. Overproduction of suppressor FlgB (but not wild-type FlgB) in the fliE mutant also resulted in improved motility. The second-site FlgB mutation by itself had no phenotype; cells swarmed as well as wild-type cells. When overproduced, wild-type FliE was dominant over FliE-V99G, but the reverse was not true; that is, overproduced FliE-V99G was not negatively dominant over wild-type FliE. We conclude that the mutant protein has reduced probability of assembly but, if assembled, functions relatively well. Export of the flagellar protein FlgD, which is known to be FliE dependent, was severely impaired by the FliE-V99G mutation but was significantly improved in the suppressor strains. The FliE mutation, V99G, was close to the C terminus of the 104-amino-acid sequence; the suppressing mutations in FlgB were all either G119E or G129D, close to the C terminus of its 138-amino-acid sequence. Affinity blotting experiments between FliE as probe and various basal body proteins as targets and vice versa revealed strong interactions between FliE and FlgB; much weaker interactions between FliE and other rod proteins were observed and probably derive from the known similarities among these proteins. We suggest that FliE subunits constitute a junction zone between the MS ring and the rod and also that the proximal rod structure consists of FlgB subunits.

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