Acylation-dependent export of Trypanosoma cruzi phosphoinositide-specific phospholipase C to the outer surface of amastigotes.
Phosphoinositide phospholipase C (PI-PLC) plays an essential role in cell signaling. A unique Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid-modified in its N terminus and localizes to the plasma membrane of amastigotes. Here, we show that TcPI-PLC is located onto the extracellular phase of the plasma membrane of amastigotes and that its N-terminal 20 amino acids are necessary and sufficient to target the fused GFP to the outer surface of the parasite. Mutagenesis of the predicted acylated residues confirmed that myristoylation of a glycine residue in the 2nd position and acyl modification of a cysteine in the 4th but not in the 8th or 15th position of the coding sequence are required for correct plasma membrane localization in T. cruzi epimastigotes or amastigotes. Interestingly, mutagenesis of the cysteine at the 8th position increased its flagellar localization. When expressed as fusion constructs with GFP, the N-terminal 6 and 10 amino acids fused to GFP are predominantly located in the cytosol and concentrated in a compartment that co-localizes with a Golgi complex marker. The N-terminal 20 amino acids of TcPI-PLC associate with lipid rafts when dually acylated. Taken together, these results indicate that N-terminal acyl modifications serve as a molecular addressing system for sending TcPI-PLC to the outer surface of the cell.
de Paulo Martins
Vicente
V
Department of Cellular Biology, University of Georgia, Center for Tropical and Emerging Global Diseases, Athens, Georgia 30602, USA.
Okura
Michael
M
Maric
Danijela
D
Engman
David M
DM
Vieira
Mauricio
M
Docampo
Roberto
R
Moreno
Silvia N J
SN
D000215:Acylation / Q000502:physiology; D000818:Animals; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D006056:Golgi Apparatus / Q000201:enzymology / Q000235:genetics; D021962:Membrane Microdomains / Q000201:enzymology* / Q000235:genetics; D016296:Mutagenesis; D054801:Phosphoinositide Phospholipase C / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D021381:Protein Transport / Q000502:physiology; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics
D015800:Protozoan Proteins; D054801:Phosphoinositide Phospholipase C