scholarly journals Clusters of Charged Residues at the C Terminus of MotA and N Terminus of MotB Are Important for Function of the Escherichia coli Flagellar Motor

2008 ◽  
Vol 190 (15) ◽  
pp. 5517-5521 ◽  
Author(s):  
Edan R. Hosking ◽  
Michael D. Manson
Keyword(s):  
Escherichia Coli ◽  
Flagellar Motor ◽  
Protein Levels ◽  
Partner Protein ◽  
C Terminus ◽  
N Terminus ◽  
Charged Residues ◽  
Positively Charged ◽  
Terminal Cluster

ABSTRACT MotA contains a conserved C-terminal cluster of negatively charged residues, and MotB contains a conserved N-terminal cluster of positively charged residues. Charge-altering mutations affecting these residues impair motility but do not diminish Mot protein levels. The motility defects are reversed by second-site mutations targeting the same or partner protein.

scholarly journals Escherichia coli l-aspartate-α-decarboxylase: preprotein processing and observation of reaction intermediates by electrospray mass spectrometry

1997 ◽  
Vol 323 (3) ◽  
pp. 661-669 ◽  
Author(s):  
Manoj K. RAMJEE ◽  
Ulrich GENSCHEL ◽  
Chris ABELL ◽  
Alison G. SMITH
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Electrospray Mass Spectrometry ◽  
Elevated Temperatures ◽  
Gene Encoding ◽  
Α Subunit ◽  
C Terminus ◽  
N Terminus ◽  
Terminal Amino ◽  
Α Subunits

The Escherichia coli panD gene, encoding l-aspartate-α-decarboxylase, was cloned by PCR, and shown to complement apanD mutant defective in β-alanine biosynthesis. Aspartate decarboxylase is a pyruvoyl-dependent enzyme, and is synthesized initially as an inactive proenzyme (the π-protein), which is proteolytically cleaved at a specific X–Ser bond to produce a β-subunit with XOH at its C-terminus and an α-subunit with a pyruvoyl group at its N-terminus, derived from the serine. The recombinant enzyme, as purified, is a tetramer, and comprises principally the unprocessed π-subunit (of 13.8 kDa), with a small proportion of the α- and β-subunits (11 kDa and 2.8 kDa respectively). Incubation of the purified enzyme at elevated temperatures for several hours results in further processing. Using fluorescein thiosemicarbazide, the completely processed enzyme was shown to contain three pyruvoyl groups per tetrameric enzyme. The presence of unchanged serine at the N-terminus of some of the α-subunits was confirmed by electrospray mass spectrometry (ESMS) and N-terminal amino acid sequencing. A novel HPLC assay for aspartate decarboxylase was established and used to determine the Km and kcat for l-aspartate as 151±16 μM and 0.57 s-1 respectively. ESMS was also used to observe substrate and product adducts trapped on the pyruvoyl group by sodium cyanoborohydride treatment.

Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation

1991 ◽  
Vol 99 (4) ◽  
pp. 823-836
Author(s):  
S.J. Atkinson ◽  
M. Stewart
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Coiled Coil ◽  
Periodic Variation ◽  
Heptad Repeat ◽  
C Terminus ◽  
N Terminus ◽  
Low Ionic Strength ◽  
Myosin Rod

We have expressed in Escherichia coli a cDNA clone corresponding broadly to rabbit light meromyosin (LMM) together with a number of modified polypeptides and have used this material to investigate the role of different aspects of molecular structure on the solubility properties of LMM. The expressed material was characterized biochemically and structurally to ensure that it retained the coiled-coil conformation of the native molecule. Full-length recombinant LMM retained the general solubility properties of myosin and, although soluble at high ionic strength, precipitated when the ionic strength was reduced below 0.3 M. Constructs in which the ‘skip’ residues (that disrupt the coiled-coil heptad repeat) were deleted had solubility properties indistinguishable from the wild type, which indicated that the skip residues did not play a major role in determining the molecular interactions involved in assembly. Deletions from the N terminus of LMM did not alter the solubility properties of the expressed material, but deletion of 92 residues from the C terminus caused a large increase in solubility at low ionic strength, indicating that a determinant important for interaction between LMM molecules was located in this region. The failure of deletions from the molecule's N terminus to alter its solubility radically suggested that the periodic variation of charge along the myosin rod may not be as important as proposed for determining the strength of binding between molecules and thus the solubility of myosin.

scholarly journals Roles of Charged Residues of Rotor and Stator in Flagellar Rotation: Comparative Study using H+-Driven and Na+-Driven Motors in Escherichia coli

2006 ◽  
Vol 188 (4) ◽  
pp. 1466-1472 ◽  
Author(s):  
Toshiharu Yakushi ◽  
Junghoon Yang ◽  
Hajime Fukuoka ◽  
Michio Homma ◽  
David F. Blair
Keyword(s):  
Escherichia Coli ◽  
Comparative Study ◽  
Electrostatic Interactions ◽  
General Feature ◽  
Flagellar Motor ◽  
Motor Patterns ◽  
E Coli ◽  
Flagellar Motors ◽  
Charged Residues ◽  
Flagellar Rotation

ABSTRACT In Escherichia coli, rotation of the flagellar motor has been shown to depend upon electrostatic interactions between charged residues of the stator protein MotA and the rotor protein FliG. These charged residues are conserved in the Na+-driven polar flagellum of Vibrio alginolyticus, but mutational studies in V. alginolyticus suggested that they are relatively unimportant for motor rotation. The electrostatic interactions detected in E. coli therefore might not be a general feature of flagellar motors, or, alternatively, the V. alginolyticus motor might rely on similar interactions but incorporate additional features that make it more robust against mutation. Here, we have carried out a comparative study of chimeric motors that were resident in E. coli but engineered to use V. alginolyticus stator components, rotor components, or both. Charged residues in the V. alginolyticus rotor and stator proteins were found to be essential for motor rotation when the proteins functioned in the setting of the E. coli motor. Patterns of synergism and suppression in rotor/stator double mutants indicate that the V. alginolyticus proteins interact in essentially the same way as their counterparts in E. coli. The robustness of the rotor-stator interface in V. alginolyticus is in part due to the presence of additional charged residues in PomA but appears mainly due to other factors, because an E. coli motor using both rotor and stator components from V. alginolyticus remained sensitive to mutation. Motor function in V. alginolyticus may be enhanced by the proteins MotX and MotY.

scholarly journals 3P201 Role of the charged residues in the rotor and stator of a Na^+-coupling flagellar motor in Escherichia coli

2005 ◽  
Vol 45 (supplement) ◽  
pp. S254
Author(s):  
Toshiharu Yakushi ◽  
Jung-Hoon Yane ◽  
Hajime Fukuoka ◽  
Michio Homma ◽  
David Blair
Keyword(s):  
Escherichia Coli ◽  
Flagellar Motor ◽  
Charged Residues

scholarly journals ANKRD22 is an N-myristoylated hairpin-like monotopic membrane protein specifically localized to lipid droplets

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Toshihiko Utsumi ◽  
Takuro Hosokawa ◽  
Mayu Shichita ◽  
Misato Nishiue ◽  
Natsuko Iwamoto ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Lipid Droplets ◽  
Transmembrane Domain ◽  
Intracellular Localization ◽  
Membrane Topology ◽  
Intracellular Targeting ◽  
C Terminus ◽  
Charged Residues ◽  
Necrosis Factor ◽  
Positively Charged

AbstractThe membrane topology and intracellular localization of ANKRD22, a novel human N-myristoylated protein with a predicted single-pass transmembrane domain that was recently reported to be overexpressed in cancer, were examined. Immunofluorescence staining of COS-1 cells transfected with cDNA encoding ANKRD22 coupled with organelle markers revealed that ANKRD22 localized specifically to lipid droplets (LD). Analysis of the intracellular localization of ANKRD22 mutants C-terminally fused to glycosylatable tumor necrosis factor (GLCTNF) and assessment of their susceptibility to protein N-glycosylation revealed that ANKRD22 is synthesized on the endoplasmic reticulum (ER) membrane as an N-myristoylated hairpin-like monotopic membrane protein with the amino- and carboxyl termini facing the cytoplasm and then sorted to LD. Pro98 located at the center of the predicted membrane domain was found to be essential for the formation of the hairpin-like monotopic topology of ANKRD22. Moreover, the hairpin-like monotopic topology, and positively charged residues located near the C-terminus were demonstrated to be required for the sorting of ANKRD22 from ER to LD. Protein N-myristoylation was found to positively affect the LD localization. Thus, multiple factors, including hairpin-like monotopic membrane topology, C-terminal positively charged residues, and protein N-myristoylation cooperatively affected the intracellular targeting of ANKRD22 to LD.

scholarly journals Expression of recombinant rat myo-inositol 1,4,5-trisphosphate 3-kinase B suggests a regulatory role for its N-terminus

1996 ◽  
Vol 319 (3) ◽  
pp. 713-716 ◽  
Author(s):  
Stephen THOMAS ◽  
Salvador SORIANO ◽  
Clive d'SANTOS ◽  
George BANTING
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Catalytic Domain ◽  
Maltose Binding Protein ◽  
Full Length ◽  
Regulatory Role ◽  
Calmodulin Binding ◽  
C Terminus ◽  
Inositol 1,4,5 Trisphosphate ◽  
N Terminus

We have expressed rat myo-inositol 1,4,5-trisphosphate (IP3) 3-kinase B as both a full-length, recombinant, non-fusion protein and a full-length, recombinant, fusion protein with maltose-binding protein (MBP) in Escherichia coli. The fusion protein with MBP is soluble, binds calmodulin and is enzymically active whereas the non-fusion protein is insoluble and does not bind calmodulin unless co-expressed with bacterial chaperone proteins (either GroES and GroEL, or DnaK, DnaJ and GrpE). However, soluble, calmodulin-binding non-fusion IP3 3-kinase B is enzymically inactive. The catalytic domain of the enzyme has previously been shown to reside near the C-terminus; the results we present suggest an auto-regulatory role for the N-terminus.

scholarly journals Puf6 and Loc1 Are the Dedicated Chaperones of Ribosomal Protein Rpl43 in Saccharomyces cerevisiae

2019 ◽  
Vol 20 (23) ◽  
pp. 5941
Author(s):  
Kai-Jen Liang ◽  
Le-Yun Yueh ◽  
Ning-Hsiang Hsu ◽  
Jui-Sheng Lai ◽  
Kai-Yin Lo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Large Subunit ◽  
Potential Interaction ◽  
Nuclear Localization Signals ◽  
Protein Levels ◽  
Trimeric Complex ◽  
C Terminus ◽  
N Terminus

Ribosomal proteins are highly expressed, and the quality of ribosomal proteins must be rigorously controlled to build up a functional ribosome. Rpl43, ribosomal protein large subunit 43, is located nearby the E-site of ribosomes. In our previous study, we found that Puf6, Loc1, and Rpl43 form a trimeric complex in Saccharomyces cerevisiae. Rpl43 protein levels are under-accumulated in the absence of PUF6 or LOC1. However, why the loss of Puf6 or Loc1 decreased the protein levels of Rpl43 remained unclear. In the present study, we further dissected the connections among these three proteins and found that the processing defects of pre-ribosomal RNA in puf6Δ and loc1Δ are similar to those of the mutant with depletion of Rpl43. The stability of newly synthesized Rpl43 protein decreased slightly in puf6Δ and significantly in loc1Δ. We also found that Puf6 and Loc1 could interact with nascent Rpl43 co-translationally via the N-terminus of Rpl43. While the association and dissociation of Rpl43 with karyopherins did not depend on Puf6 and Loc1, Puf6 and Loc1 interacted with nascent Rpl43 in collaboration. While the N-terminus of Puf6 contained nuclear localization signals for transport, the PUF (Pumilio) domain was essential to interaction with Loc1, Rpl43, and 60S subunits. The C-terminus of Loc1 is more important for interaction with Puf6 and Rpl43. In this study, we found that Puf6 and Loc1 are the dedicated chaperones of ribosomal protein Rpl43 and also analyzed the potential interaction domains among the three proteins. Correct formation of the Puf6, Loc1, and Rpl43 ternary complex is required to properly proceed to the next step in 60S biogenesis.

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