Dual acylation accounts for the localization of {alpha}19-giardin in the ventral flagellum pair of Giardia lamblia.
A Giardia-specific protein family denominated as alpha-giardins, represents the major protein component, besides tubulin, of the cytoskeleton of the human pathogenic parasite Giardia lamblia. One of its members, alpha19-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 alpha19-giardin to be associated with the membrane fraction of disrupted trophozoites. After heterologous coexpression of alpha19-giardin with giardial N-myristoyltransferase (NMT) in Escherichia coli, we found the protein in a myristoylated form. Additionally, after heterologous expression together with the palmitoyl transferase Pfa3 in Saccharomyces cerevisiae, alpha19-giardin associates with the membrane of the main vacuole. Immunocytochemical colocalization studies on wild-type Giardia trophozoites with tubulin provide evidence that alpha19-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 alpha19-giardin is responsible for its specific flagellar localization.
Saric
Mirela
M
Faculty of Biology/Chemistry, Barbarastrasse 13, D-49069 Osnabrueck, Germany.
Vahrmann
Anke
A
Niebur
Daniela
D
Kluempers
Verena
V
Hehl
Adrian B
AB
Scholze
Henning
H
D000215:Acylation; D003598:Cytoskeletal Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D005407:Flagella / Q000378:metabolism*; D016829:Giardia lamblia / Q000378:metabolism*; D018613:Microscopy, Confocal; D015800:Protozoan Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism
D003598:Cytoskeletal Proteins; D015800:Protozoan Proteins; C038207:giardin protein, Giardia lamblia
J Cell Sci. 2000 Jul;113 ( Pt 14):2607-18
10862718
Mol Microbiol. 2005 Nov;58(4):999-1011
16262786
Biochemistry. 2006 Feb 21;45(7):2339-49
16475823
J Biol Chem. 2001 Oct 26;276(43):39501-4
11527981
Electrophoresis. 1988 Jun;9(6):255-62
2466658
Science. 1989 Jan 6;243(4887):75-7
2911720
EMBO J. 1998 Jun 15;17(12):3269-76
9628864
Methods Enzymol. 1979;56:58-66
379518
Int J Parasitol. 2008 Sep;38(11):1297-304
18625508
Eukaryot Cell. 2006 Apr;5(4):753-61
16607022
Traffic. 2009 Aug;10(8):1061-73
19453970
Biochem J. 1993 Apr 15;291 ( Pt 2):601-8
8484740
EMBO J. 1999 Apr 15;18(8):2057-65
10205160
Biochem J. 1997 Feb 15;322 ( Pt 1):49-56
9078242
Parasitol Today. 1990 Feb;6(2):52-6
15463296
Mol Biol Cell. 2003 Apr;14(4):1433-47
12686599
J Mol Biol. 2009 Jan 30;385(4):1098-112
19046974
Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5519-23
8516294
J Mol Biol. 2002 Apr 5;317(4):541-57
11955008
Science. 2007 Sep 28;317(5846):1921-6
17901334
Annu Rev Microbiol. 1996;50:679-705
8905095
J Biol Chem. 1987 Jul 25;262(21):10035-8
3611052
J Mol Biol. 2007 Apr 27;368(2):493-508
17355882
J Biol Chem. 1994 Jan 28;269(4):2996-3009
8300631
Proc Natl Acad Sci U S A. 2005 Nov 29;102(48):17366-71
16301533
J Biol Chem. 2002 Jul 12;277(28):25703-6
12006598
Curr Genet. 1993 Nov;24(5):373-6
8299150
FEMS Microbiol Lett. 1999 Apr 1;173(1):147-53
10220891
Trends Cell Biol. 2003 Jan;13(1):32-42
12480338
Cell Signal. 1996 Sep;8(6):403-12
8958442
J Exp Biol. 1974 Feb;60(1):207-21
4594683
Mol Biol Cell. 2008 Jul;19(7):3124-37
18463165
Trans R Soc Trop Med Hyg. 1983;77(4):487-8
6636276
Eukaryot Cell. 2008 Apr;7(4):569-74
18296622
J Cell Sci. 2006 Jun 15;119(Pt 12):2477-85
16720644
J Mol Evol. 1997 Feb;44(2):178-88
9069178
Int J Parasitol. 2005 May;35(6):617-26
15862575
Mol Biol Cell. 2000 May;11(5):1789-800
10793152
Infect Immun. 2001 Dec;69(12):7866-72
11705969
Mol Biol Evol. 1995 Nov;12(6):967-79
8524049
Protein Expr Purif. 1994 Apr;5(2):133-7
8054844
Int J Parasitol. 2003 Jan;33(1):3-28
12547343
Parasitol Res. 2008 Jan;102(2):321-6
17938962
FEMS Microbiol Lett. 2000 Aug 15;189(2):271-3
10930750
Physiol Rev. 2002 Apr;82(2):331-71
11917092