Targeting of the GRIP domain to the trans-Golgi network is conserved from protists to animals.
The GRIP domain, found in a family of coiled-coil peripheral membrane Golgi proteins, is a specific targeting sequence for the trans-Golgi network of animal cells. In this study we show that a coiled-coil protein with a GRIP domain occurs in the primitive eukaryote, Trypanosoma brucei, and that reporter proteins containing this domain can be used as a marker for the poorly characterized trans Golgi/trans-Golgi network of trypanosomatid parasites. The T. brucei GRIP domain, when fused to the carboxyl terminus of the green fluorescent protein (GFP-TbGRIP), was efficiently localized to the Golgi apparatus of transfected COS cells. Overexpression of GFP-TbGRIP in COS cells displaced the endogenous GRIP protein, GCC1p, from the Golgi apparatus indicating that the trypanosomatid and mammalian GRIP sequences interact with similar membrane determinants. GFP fusion proteins containing either the T. brucei GRIP domain or the human p230 GRIP (p230GRIP) domain were also expressed in the trypanosomatid parasite, Leishmania mexicana, and localized by fluorescence and immuno-electron microscopy to the trans face of the single Golgi apparatus and a short tubule that extended from the Golgi apparatus. Binding of GFP-p230GRIP to Golgi membranes in L. mexicana was abrogated by mutation of a critical tyrosine residue in the p230 GRIP domain. The levels of GFP-GRIP fusion proteins were dramatically reduced in stationary-phase L. mexicana promastigotes, suggesting that specific Golgi trafficking steps may be down-regulated as the promastigotes cease dividing. This study provides a protein marker for the trans-Golgi network of trypanosomatid parasites and suggests that the GRIP domain binds to a membrane component that has been highly conserved in eukaryotic evolution.
McConville
Malcolm J
MJ
Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Victoria, Australia.
Ilgoutz
Steven C
SC
Teasdale
Rohan D
RD
Foth
Bernardo J
BJ
Matthews
Antony
A
Mullin
Kylie A
KA
Gleeson
Paul A
PA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D017124:Conserved Sequence; D015536:Down-Regulation; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D007894:Leishmania mexicana / Q000378:metabolism / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D014346:Trypanosoma brucei brucei / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism* / Q000648:ultrastructure
D021382:Protein Sorting Signals