A novel functional element in the N-terminal region of Arum concinnatum alternative oxidase is indispensable for catalytic activity of the enzyme in HeLa cells

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.

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Identification of the Helicobacter pylori VacA Toxin Domain Active in the Cell Cytosol

Infection and Immunity ◽

10.1128/iai.66.12.6014-6016.1998 ◽

1998 ◽

Vol 66 (12) ◽

pp. 6014-6016 ◽

Cited By ~ 75

Author(s):

Marina de Bernard ◽

Daniela Burroni ◽

Emanuele Papini ◽

Rino Rappuoli ◽

John Telford ◽

...

Keyword(s):

Helicobacter Pylori ◽

Hela Cells ◽

Terminal Region ◽

Endocytic Pathway ◽

Amino Terminal ◽

Toxin Molecule ◽

Intracellular Activity ◽

Cell Cytosol ◽

C And N ◽

Late Stages

ABSTRACT Cells exposed to Helicobacter pylori toxin VacA develop large vacuoles which originate from massive swelling of membranous compartments at late stages of the endocytic pathway. When expressed in the cytosol, VacA induces vacuolization as it does when added from outside. This and other evidence indicate that VacA is a toxin capable of entering the cell cytosol, where it displays its activity. In this study, we have used cytosolic expression to identify the portion of the toxin molecule responsible for the vacuolating activity. VacA mutants with deletions at the C and N termini were generated, and their activity was analyzed upon expression in HeLa cells. We found that the vacuolating activity of VacA resides in the amino-terminal region, the whole of which is required for its intracellular activity.

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EPI64 interacts with Slp1/JFC1 to coordinate Rab8a and Arf6 membrane trafficking

Molecular Biology of the Cell ◽

10.1091/mbc.e11-06-0521 ◽

2012 ◽

Vol 23 (4) ◽

pp. 701-715 ◽

Cited By ~ 17

Author(s):

David E. Hokanson ◽

Anthony P. Bretscher

Keyword(s):

Hela Cells ◽

Membrane Trafficking ◽

Binding Proteins ◽

Cell Function ◽

Terminal Region ◽

Cytoskeletal Organization ◽

Vacuole Formation ◽

Gtp Binding ◽

Distinctive Phenotype ◽

In Cells

Cell function requires the integration of cytoskeletal organization and membrane trafficking. Small GTP-binding proteins are key regulators of these processes. We find that EPI64, an apical microvillar protein with a Tre-2/Bub2/Cdc16 (TBC) domain that stabilizes active Arf6 and has RabGAP activity, regulates Arf6-dependent membrane trafficking. Expression of EPI64 in HeLa cells induces the accumulation of actin-coated vacuoles, a distinctive phenotype seen in cells expressing constitutively active Arf6. Expression of EPI64 with defective RabGAP activity does not induce vacuole formation. Coexpression of Rab8a suppresses the vacuole phenotype induced by EPI64, and EPI64 expression lowers the level of Rab8-GTP in cells, strongly suggesting that EPI64 has GAP activity toward Rab8a. JFC1, an effector for Rab8a, colocalizes with and binds directly to a C-terminal region of EPI64. Together this region and the N-terminal TBC domain of EPI64 are required for the accumulation of vacuoles. Through analysis of mutants that uncouple JFC1 from either EPI64 or from Rab8-GTP, our data suggest a model in which EPI64 binds JFC1 to recruit Rab8a-GTP for deactivation by the RabGAP activity of EPI64. We propose that EPI64 regulates membrane trafficking both by stabilizing Arf6-GTP and by inhibiting the recycling of membrane through the tubular endosome by decreasing Rab8a-GTP levels.

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Identification of a binding element for the cytoplasmic regulator FROUNT in the membrane-proximal C-terminal region of chemokine receptors CCR2 and CCR5

Biochemical Journal ◽

10.1042/bj20130827 ◽

2013 ◽

Vol 457 (2) ◽

pp. 313-322 ◽

Cited By ~ 7

Author(s):

Etsuko Toda ◽

Yuya Terashima ◽

Kaori Esaki ◽

Sosuke Yoshinaga ◽

Minoru Sugihara ◽

...

Keyword(s):

Chemokine Receptors ◽

Functional Element ◽

Terminal Region ◽

Regulatory Mechanisms

FROUNT promotes chemotaxis by interacting with chemokine receptors via unknown mechanisms. In the present study, we identified a functional element crucial for FROUNT binding at the GPCR helix 8 region. This finding will facilitate better understanding of GPCR regulatory mechanisms via cytoplasmic regulators.

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