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Eukaryotic Cell, February 2009, p. 190-196, Vol. 8, No. 2
1535-9778/09/$08.00+0     doi:10.1128/EC.00201-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

GAP45 Phosphorylation Controls Assembly of the Toxoplasma Myosin XIV Complex{triangledown}

Stacey D. Gilk,1,{dagger} Elizabeth Gaskins,1 Gary E. Ward,2 and Con J. M. Beckers1*

Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina 27599,1 Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 054052

Received 18 June 2008/ Accepted 20 November 2008

Toxoplasma gondii motility is powered by the myosin XIV motor complex, which consists of the myosin XIV heavy chain (MyoA), the myosin light chain (MLC1), GAP45, and GAP50, the membrane anchor of the complex. MyoA, MLC1, and GAP45 are initially assembled into a soluble complex, which then associates with GAP50, an integral membrane protein of the parasite inner membrane complex. While all proteins in the myosin XIV motor complex are essential for parasite survival, the specific role of GAP45 remains unclear. We demonstrate here that final assembly of the motor complex is controlled by phosphorylation of GAP45. This protein is phosphorylated on multiple residues, and by using mass spectroscopy, we have identified two of these, Ser163 and Ser167. The importance of these phosphorylation events was determined by mutation of Ser163 and Ser167 to Glu and Ala residues to mimic phosphorylated and nonphosphorylated residues, respectively. Mutation of Ser163 and Ser167 to either Ala or Glu residues does not affect targeting of GAP45 to the inner membrane complex or its association with MyoA and MLC1. Mutation of Ser163 and Ser167 to Ala residues also does not affect assembly of the mutant GAP45 protein into the myosin motor complex. Mutation of Ser163 and Ser167 to Glu residues, however, prevents association of the MyoA-MLC1-GAP45 complex with GAP50. These observations indicate that phosphorylation of Ser163 and Ser167 in GAP45 controls the final step in assembly of the myosin XIV motor complex.

* Corresponding author. Mailing address: Department of Cell and Developmental Biology, 108 Taylor Hall, University of North Carolina, Chapel Hill, NC 27599. Phone: (919) 966-1464. Fax: (919) 966-1856. E-mail: cbeckers{at}med.unc.edu

{triangledown} Published ahead of print on 1 December 2008.

{dagger} Present address: Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, NIAID/NIH, Hamilton, MT 59840.

Eukaryotic Cell, February 2009, p. 190-196, Vol. 8, No. 2
1535-9778/09/$08.00+0     doi:10.1128/EC.00201-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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