scholarly journals Dual Acylation Accounts for the Localization of α19-Giardin in the Ventral Flagellum Pair of Giardia lamblia

2009 ◽  
Vol 8 (10) ◽  
pp. 1567-1574 ◽  
Author(s):  
Mirela Šarić ◽  
Anke Vahrmann ◽  
Daniela Niebur ◽  
Verena Kluempers ◽  
Adrian B. Hehl ◽  
...  
Keyword(s):  
Giardia Lamblia ◽  
Specific Protein ◽  
Cysteine Residue ◽  
Major Protein ◽  
Lipid Modification ◽  
Wild Type ◽  
Terminal Sequence ◽  
Glycine Residue ◽  
Specific Localization ◽  
Pathogenic Parasite

ABSTRACT A Giardia-specific protein family denominated as α-giardins, represents the major protein component, besides tubulin, of the cytoskeleton of the human pathogenic parasite Giardia lamblia. One of its members, α19-giardin, carries an N-terminal sequence extension of MGCXXS, which in many proteins serves as a target for dual lipid conjugation: myristoylation at the glycine residue after removal of the methionine and palmitoylation at the cysteine residue. As the first experimental evidence of a lipid modification, we found α19-giardin to be associated with the membrane fraction of disrupted trophozoites. After heterologous coexpression of α19-giardin with giardial N-myristoyltransferase (NMT) in E scherichia coli, we found the protein in a myristoylated form. Additionally, after heterologous expression together with the palmitoyl transferase Pfa3 in Saccharomyces cerevisiae, α19-giardin associates with the membrane of the main vacuole. Immunocytochemical colocalization studies on wild-type Giardia trophozoites with tubulin provide evidence that α19-giardin exclusively localizes to the ventral pair of the giardial flagella. A mutant in which the putatively myristoylated N-terminal glycine residue was replaced by alanine lost this specific localization. Our findings suggest that the dual lipidation of α19-giardin is responsible for its specific flagellar localization.

scholarly journals INTRACISTRONIC MAPPING OF ELECTROPHORETIC SITES IN DROSOPHILA MELANOGASTER: FIDELITY OF INFORMATION TRANSFER BY GENE CONVERSION

Genetics ◽  
1974 ◽  
Vol 76 (2) ◽  
pp. 289-299
Author(s):  
Margaret McCarron ◽  
William Gelbart ◽  
Arthur Chovnick
Keyword(s):  
Drosophila Melanogaster ◽  
Information Transfer ◽  
Large Scale ◽  
Genetic Basis ◽  
Xanthine Dehydrogenase ◽  
Specific Protein ◽  
Wild Type ◽  
Electrophoretic Variation ◽  
The Stability ◽  
Recombination Experiments

ABSTRACT A convenient method is described for the intracistronic mapping of genetic sites responsible for electrophoretic variation of a specific protein in Drosophila melanogaster. A number of wild-type isoalleles of the rosy locus have been isolated which are associated with the production of electrophoretically distinguishable xanthine dehydrogenases. Large-scale recombination experiments were carried out involving null enzyme mutants induced on electrophoretically distinct wild-type isoalleles, the genetic basis for which is followed as a nonselective marker in the cross. Additionally, a large-scale recombination experiment was carried out involving null enzyme rosy mutants induced on the same wild-type isoallele. Examination of the electrophoretic character of crossover and convertant products recovered from the latter experiment revealed that all exhibited the same parental electrophoretic character. In addition to documenting the stability of the xanthine dehydrogenase electrophoretic character, this observation argues against a special mutagenesis hypothesis to explain conversions resulting from allele recombination studies.

scholarly journals Synapsin I (protein I), a nerve terminal-specific phosphoprotein. I. Its general distribution in synapses of the central and peripheral nervous system demonstrated by immunofluorescence in frozen and plastic sections.

1983 ◽  
Vol 96 (5) ◽  
pp. 1337-1354 ◽  
Author(s):  
P De Camilli ◽  
R Cameron ◽  
P Greengard
Keyword(s):  
Nervous System ◽  
Detailed Examination ◽  
Specific Protein ◽  
Nerve Cells ◽  
General Distribution ◽  
Synapsin I ◽  
Nervous Systems ◽  
Synaptic Region ◽  
Specific Localization ◽  
Peripheral Nervous Systems

Synapsin I (formerly referred to as protein I) is the collective name for two almost identical phosphoproteins, synapsin Ia and synapsin Ib (protein Ia and protein Ib), present in the nervous system. Synapsin I has previously been shown by immunoperoxidase studies (De Camilli, P., T. Ueda, F. E. Bloom, E. Battenberg, and P. Greengard, 1979, Proc. Natl. Acad. Sci. USA, 76:5977-5981; Bloom, F. E., T. Ueda, E. Battenberg, and P. Greengard, 1979, Proc. Natl. Acad. Sci. USA 76:5982-5986) to be a neuron-specific protein, present in both the central and peripheral nervous systems and concentrated in the synaptic region of nerve cells. In those preliminary studies, the occurrence of synapsin I could be demonstrated in only a portion of synapses. We have now carried out a detailed examination of the distribution of synapsin I immunoreactivity in the central and peripheral nervous systems. In this study we have attempted to maximize the level of resolution of immunohistochemical light microscopy images in order to estimate the proportion of immunoreactive synapses and to establish their precise distribution. Optimal results were obtained by the use of immunofluorescence in semithin sections (approximately 1 micron) prepared from Epon-embedded nonosmicated tissues after the Epon had been removed. Our results confirm the previous observations on the specific localization of synapsin I in nerve cells and synapses. In addition, the results strongly suggest that, with a few possible exceptions involving highly specialized neurons, all synapses contain synapsin I. Finally, immunocytochemical experiments indicate that synapsin I appearance in the various regions of the developing nervous system correlates topographically and temporally with the appearance of synapses. In two accompanying papers (De Camilli, P., S. M. Harris, Jr., W. B. Huttner, and P. Greengard, and Huttner, W. B., W. Schiebler, P. Greengard, and P. De Camilli, 1983, J. Cell Biol. 96:1355-1373 and 1374-1388, respectively), evidence is presented that synapsin I is specifically associated with synaptic vesicles in nerve endings.

scholarly journals Insights on the mechanisms of Ca2+ regulation of connexin26 hemichannels revealed by human pathogenic mutations (D50N/Y)

2013 ◽  
Vol 142 (1) ◽  
pp. 23-35 ◽  
Author(s):  
William Lopez ◽  
Jorge Gonzalez ◽  
Yu Liu ◽  
Andrew L. Harris ◽  
Jorge E. Contreras
Keyword(s):  
Essential Role ◽  
Negative Charge ◽  
Salt Bridge ◽  
Cysteine Residue ◽  
Wild Type ◽  
Closed State ◽  
Large Size ◽  
Deactivation Kinetics ◽  
Connexin Hemichannel

Because of the large size and modest selectivity of the connexin hemichannel aqueous pore, hemichannel opening must be highly regulated to maintain cell viability. At normal resting potentials, this regulation is achieved predominantly by the physiological extracellular Ca2+ concentration, which drastically reduces hemichannel activity. Here, we characterize the Ca2+ regulation of channels formed by wild-type human connexin26 (hCx26) and its human mutations, D50N/Y, that cause aberrant hemichannel opening and result in deafness and skin disorders. We found that in hCx26 wild-type channels, deactivation kinetics are accelerated as a function of Ca2+ concentration, indicating that Ca2+ facilitates transition to, and stabilizes, the closed state of the hemichannels. The D50N/Y mutant hemichannels show lower apparent affinities for Ca2+-induced closing than wild-type channels and have more rapid deactivation kinetics, which are Ca2+ insensitive. These results suggest that D50 plays a role in (a) stabilizing the open state in the absence of Ca2+, and (b) facilitating closing and stabilization of the closed state in the presence of Ca2+. To explore the role of a negatively charged residue at position 50 in regulation by Ca2+, this position was substituted with a cysteine residue, which was then modified with a negatively charged methanethiosulfonate reagent, sodium (2-sulfanoethyl) methanethiosulfonate (MTSES)−. D50C mutant hemichannels display properties similar to those of D50N/Y mutants. Recovery of the negative charge with chemical modification by MTSES− restores the wild-type Ca2+ regulation of the channels. These results confirm the essential role of a negative charge at position 50 for Ca2+ regulation. Additionally, charge-swapping mutagenesis studies suggest involvement of a salt bridge interaction between D50 and K61 in the adjacent connexin subunit in stabilizing the open state in low extracellular Ca2+. Mutant cycle analysis supports a Ca2+-sensitive interaction between these two residues in the open state of the channel. We propose that disruption of this interaction by extracellular Ca2+ destabilizes the open state and facilitates hemichannel closing. Our data provide a mechanistic understanding of how mutations at position 50 that cause human diseases are linked to dysfunction of hemichannel gating by external Ca2+.

scholarly journals Palmitoylation of the Autographa californica Multicapsid Nucleopolyhedrovirus Envelope Glycoprotein GP64: Mapping, Functional Studies, and Lipid Rafts

2003 ◽  
Vol 77 (11) ◽  
pp. 6265-6273 ◽  
Author(s):  
Sandy Xiaoxin Zhang ◽  
Yu Han ◽  
Gary W. Blissard
Keyword(s):  
Cell Surface ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Envelope Glycoprotein ◽  
Cysteine Residue ◽  
Autographa Californica ◽  
Membrane Microdomains ◽  
Sf9 Cells ◽  
Wild Type ◽  
Lipid Raft Microdomains

ABSTRACT Budded virions (BV) of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) contain a major envelope glycoprotein known as GP64, which was previously shown to be palmitoylated. In the present study, we used truncation and amino acid substitution mutations to map the palmitoylation site to cysteine residue 503. Palmitoylation of GP64 was not detected when Cys503 was replaced with alanine or serine. Palmitoylation-minus forms of GP64 were used to replace wild-type GP64 in AcMNPV, and these viruses were used to examine potential functions of GP64 palmitoylation in the context of the infection cycle. Analysis by immunoprecipitation and cell surface studies revealed that palmitoylation of GP64 did not affect GP64 synthesis or its transport to the cell surface in Sf9 cells. GP64 proteins lacking palmitoylation also mediated low-pH-triggered membrane fusion in a manner indistinguishable from that of wild-type GP64. Cells infected with viruses expressing palmitoylation-minus forms of GP64 produced infectious virions at levels similar to those from cells infected with wild-type AcMNPV. In combination, these data suggest that virus entry and exit in Sf9 cells were not significantly affected by GP64 palmitoylation. To determine whether GP64 palmitoylation affected the association of GP64 with membrane microdomains, the potential association of GP64 with lipid raft microdomains was examined. These experiments showed that: (i) AcMNPV-infected Sf9 cell membranes contain lipid raft microdomains, (ii) GP64 association with lipid rafts was not detected in infected Sf9 cells, and (iii) GP64 palmitoylation did not affect the apparent exclusion of GP64 from lipid raft microdomains.

Farnesyl cysteine C-terminal methyltransferase activity is dependent upon the STE14 gene product in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (10) ◽  
pp. 5071-5076
Author(s):  
C A Hrycyna ◽  
S Clarke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cysteine Residue ◽  
Peptide Sequence ◽  
Direct Result ◽  
Wild Type ◽  
Methyltransferase Activity ◽  
Ras Protein ◽  
Carboxyl Methyltransferase ◽  
C Terminus ◽  
Mutant Cells

Membrane extracts of sterile Saccharomyces cerevisiae strains containing the a-specific ste14 mutation lack a farnesyl cysteine C-terminal carboxyl methyltransferase activity that is present in wild-type a and alpha cells. Other a-specific sterile strains with ste6 and ste16 mutations also have wild-type levels of the farnesyl cysteine carboxyl methyltransferase activity. This enzyme activity, detected by using a synthetic peptide sequence based on the C-terminus of a ras protein, may be responsible not only for the essential methylation of the farnesyl cysteine residue of a mating factor, but also for the methylation of yeast RAS1 and RAS2 proteins and possibly other polypeptides with similar C-terminal structures. We demonstrate that the farnesylation of the cysteine residue in the peptide is required for the methyltransferase activity, suggesting that methyl esterification follows the lipidation reaction in the cell. To show that the loss of methyltransferase activity is a direct result of the ste14 mutation, we transformed ste14 mutant cells with a plasmid complementing the mating defect of this strain and found that active enzyme was produced. Finally, we demonstrated that a similar transformation of cells possessing the wild-type STE14 gene resulted in sixfold overproduction of the enzyme. Although more complicated possibilities cannot be ruled out, these results suggest that STE14 is a candidate for the structural gene for a methyltransferase involved in the formation of isoprenylated cysteine alpha-methyl ester C-terminal structures.

Phenotypic rescue of mutant brown melanocytes by a retrovirus carrying a wild-type tyrosinase-related protein gene

Development ◽  
1990 ◽  
Vol 110 (2) ◽  
pp. 471-475 ◽  
Author(s):  
D.C. Bennett ◽  
D. Huszar ◽  
P.J. Laipis ◽  
R. Jaenisch ◽  
I.J. Jackson
Keyword(s):  
Coat Colour ◽  
Specific Protein ◽  
Related Protein ◽  
Histone H4 ◽  
Wild Type ◽  
Leukaemia Virus ◽  
Mouse Cdna ◽  
Phenotypic Rescue ◽  
Moloney Murine Leukaemia Virus ◽  
Tyrosinase Related Protein

A mouse cDNA for the developmentally controlled, melanocyte-specific protein, tyrosinase-related protein 1 (TRP-1), was previously cloned and reported to show genetic linkage with the coat-colour locus brown (b) on mouse chromosome 4. The cDNA has been inserted into a retroviral vector derived from Moloney murine leukaemia virus, under the control of the human histone H4 promoter. This vector was used to infect melanocytes of the immortal line melan-b, which are homozygous for the b mutation and which display light brown pigmentation in culture. Infected cultures containing between 0.2 and 2 copies of provirus per cell displayed an altered phenotype: 20–50% of cells now had the black to dark brown colour characteristic of cultured wild-type (Black, B/B) mouse melanocytes. Thus the TRP-1 gene complements the brown mutation. We conclude that TRP-1 is the product of the wild-type b-locus.

scholarly journals Molecular Alterations in the Stomach of Tff1-Deficient Mice: Early Steps in Antral Carcinogenesis

2020 ◽  
Vol 21 (2) ◽  
pp. 644 ◽  
Author(s):  
Eva B. Znalesniak ◽  
Franz Salm ◽  
Werner Hoffmann
Keyword(s):  
Cysteine Residue ◽  
Molecular Forms ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Rt Pcr ◽  
Molecular Alterations ◽  
Protein Levels ◽  
Increased Sensitivity ◽  
A Minor ◽  
Deficient Mice

TFF1 is a peptide of the gastric mucosa co-secreted with the mucin MUC5AC. It plays a key role in gastric mucosal protection and repair. Tff1-deficient (Tff1KO) mice obligatorily develop antropyloric adenoma and about 30% progress to carcinomas. Thus, these mice represent a model for gastric tumorigenesis. Here, we compared the expression of selected genes in Tff1KO mice and the corresponding wild-type animals (RT-PCR analyses). Furthermore, we systematically investigated the different molecular forms of Tff1 and its heterodimer partner gastrokine-2 (Gkn2) in the stomach (Western blot analyses). As a hallmark, a large portion of murine Tff1 occurs in a monomeric form. This is unexpected because of its odd number of seven cysteine residues. Probably the three conserved acid amino acid residues (EEE) flanking the 7th cysteine residue allow monomeric secretion. As a consequence, the free thiol of monomeric Tff1 could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. Furthermore, a minor subset of Tff1 forms a disulfide-linked heterodimer with IgG Fc binding protein (Fcgbp). Of special note, in Tff1KO animals a homodimeric form of Gkn2 was observed. In addition, Tff1KO animals showed strongly reduced Tff2 transcript and protein levels, which might explain their increased sensitivity to Helicobacter pylori infection.

Increased susceptibility to fatigue of slow- and fast-twitch muscles from mice lacking the MG29 gene

2000 ◽  
Vol 4 (1) ◽  
pp. 43-49 ◽  
Author(s):  
RAMAKRISHNAN Y. NAGARAJ ◽  
CHRISTOPHER M. NOSEK ◽  
MARCO A. P. BROTTO ◽  
MIYUKI NISHI ◽  
HIROSHI TAKESHIMA ◽  
...  
Keyword(s):  
Major Protein ◽  
Force Frequency ◽  
Wild Type ◽  
Membrane Structures ◽  
Intracellular Ca ◽  
Slow Twitch Muscle ◽  
Fast Twitch Muscle ◽  
Frequency Relationship ◽  
Fast Twitch

Mitsugumin 29 (MG29), a major protein component of the triad junction in skeletal muscle, has been identified to play roles in the formation of precise junctional membrane structures important for efficient signal conversion in excitation-contraction (E-C) coupling. We carried out several experiments to not only study the role of MG29 in normal muscle contraction but also to determine its role in muscle fatigue. We compared the in vitro contractile properties of three muscles types, extensor digitorum longus (EDL) (fast-twitch muscle), soleus (SOL) (slow-twitch muscle), and diaphragm (DPH) (mixed-fiber muscle), isolated from mice lacking the MG29 gene and wild-type mice prior to and after fatigue. Our results indicate that the mutant EDL and SOL muscles, but not DPH, are more susceptible to fatigue than the wild-type muscles. The mutant muscles not only fatigued to a greater extent but also recovered significantly less than the wild-type muscles. Following fatigue, the mutant EDL and SOL muscles produced lower twitch forces than the wild-type muscles; in addition, fatiguing produced a downward shift in the force-frequency relationship in the mutant mice compared with the wild-type controls. Our results indicate that fatiguing affects the E-C components of the mutant EDL and SOL muscles, and the effect of fatigue in these mutant muscles could be primarily due to an alteration in the intracellular Ca homeostasis.

scholarly journals Cysteine Is Exported from theEscherichia coliCytoplasm by CydDC, an ATP-binding Cassette-type Transporter Required for Cytochrome Assembly

2002 ◽  
Vol 277 (51) ◽  
pp. 49841-49849 ◽  
Author(s):  
Marc S. Pittman ◽  
Hazel Corker ◽  
Guanghui Wu ◽  
Marie B. Binet ◽  
Arthur J. G. Moir ◽  
...  
Keyword(s):  
Redox Homeostasis ◽  
Membrane Vesicles ◽  
Major Facilitator Superfamily ◽  
Atp Binding ◽  
Major Protein ◽  
Wild Type ◽  
Independent Manner ◽  
Periplasmic Transport ◽  
Major Facilitator ◽  
Atp Binding Cassette

Assembly ofEscherichia colicytochromebdand periplasmic cytochromes requires the ATP-binding cassette transporter CydDC, whose substrate is unknown. Two-dimensional SDS-PAGE comparison of periplasm from wild-type andcydDmutant strains revealed that the latter was deficient in several periplasmic transport binding proteins, but no single major protein was missing in thecydDperiplasm. Instead, CydDC exports from cytoplasm to periplasm the amino acid cysteine, demonstrated using everted membrane vesicles that transported radiolabeled cysteine inward in an ATP-dependent, uncoupler-independent manner. New pleiotropiccydDphenotypes are reported, including sensitivity to benzylpenicillin and dithiothreitol, and loss of motility, consistent with periplasmic defects in disulfide bond formation. Exogenous cysteine reversed these phenotypes and affected levels of periplasmicc-type cytochromes incydDand wild-type strains but did not restore cytochromed. Consistent with CydDC being a cysteine exporter,cydDmutant growth was hypersensitive to high cysteine concentrations and accumulated higher cytoplasmic cysteine levels, as did a mutant defective inorf299, encoding a transporter of the major facilitator superfamily. AcydD orf299double mutant was extremely cysteine-sensitive and had higher cytoplasmic cysteine levels, whereas CydDC overexpression conferred resistance to high extracellular cysteine concentrations. We propose that CydDC exports cysteine, crucial for redox homeostasis in the periplasm.

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