HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 2, 1075-1085, January 10, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/2/1075    most recent M209436200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Drozdowicz, Y. M. Articles by Rea, P. A. Search for Related Content PubMed PubMed Citation Articles by Drozdowicz, Y. M. Articles by Rea, P. A. Social Bookmarking                      What's this?

Isolation and Characterization of TgVP1, a Type I Vacuolar H⁺-translocating Pyrophosphatase from Toxoplasma gondii
THE DYNAMICS OF ITS SUBCELLULAR LOCALIZATION AND THE CELLULAR EFFECTS OF A DIPHOSPHONATE INHIBITOR^*

Yolanda M. Drozdowicz, Michael Shaw, Manami Nishi, Boris Striepen, Helene A. Liwinski, David S. Roos, and Philip A. Rea^§

From the Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Here we report the isolation and characterization of a type I vacuolar-type H⁺-pyrophosphatase (V-PPase), TgVP1, from an apicomplexan, Toxoplasma gondii, a parasitic protist that is particularly amenable to molecular and genetic manipulation. The 816-amino acid TgVP1 polypeptide is 50% sequence-identical (65% similar) to the prototypical type I V-PPase from Arabidopsis thaliana, AVP1, and contains all the sequence motifs characteristic of this pump category. Unlike AVP1 and other known type I enzymes, however, TgVP1 contains a 74-residue N-terminal extension encompassing a 42-residue N-terminal signal peptide sequence, sufficient for targeting proteins to the secretory pathway of T. gondii. Providing that the coding sequence for the entire N-terminal extension is omitted from the plasmid, transformation of Saccharomyces cerevisiae with plasmid-borne TgVP1 yields a stable and functional translation product that is competent in aminomethylenediphosphonate (AMDP)-inhibitable K⁺-activated pyrophosphate (PP_i) hydrolysis and PP_i-energized H⁺ translocation. Immunofluorescence microscopy of both free and intracellular T. gondii tachyzoites using purified universal V-PPase polyclonal antibodies reveals a punctate apical distribution for the enzyme. Equivalent studies of the tachyzoites during host cell invasion, by contrast, disclose a transverse radial distribution in which the V-PPase is associated with a collar-like structure that migrates along the length of the parasite in synchrony with and in close apposition to the penetration furrow. Although treatment of T. gondii with AMDP concentrations as high as 100 µM had no discernible effect on the efficiency of host cell invasion and integration, concentrations commensurate with the I₅₀ for the inhibition of TgVP1 activity in vitro (0.9 µM) do inhibit cell division and elicit nuclear enlargement concomitant with the inflation and eventual disintegration of acidocalcisome-like vesicular structures. A dynamic association of TgVP1 with the host cell invasion apparatus is invoked, one in which the effects of inhibitory V-PPase substrate analogs are exerted after rather than during host cell invasion.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. M. Harper, M.-H. Huynh, I. Coppens, F. Parussini, S. Moreno, and V. B. Carruthers A Cleavable Propeptide Influences Toxoplasma Infection by Facilitating the Trafficking and Secretion of the TgMIC2-M2AP Invasion Complex Mol. Biol. Cell, October 1, 2006; 17(10): 4551 - 4563. [Abstract] [Full Text] [PDF]

				K. M. Au, R. D. Barabote, K. Y. Hu, and M. H. Saier Jr Evolutionary appearance of H+-translocating pyrophosphatases. Microbiology, May 1, 2006; 152(Pt 5): 1243 - 1247. [Full Text] [PDF]