The effector domain of Rab6, plus a highly hydrophobic C terminus, is required for Golgi apparatus localization.
C-terminal lipid modifications are essential for the interaction of Ras-related proteins with membranes. While all Ras proteins are farnesylated and some palmitoylated, the majority of other Ras-related proteins are geranylgeranylated. One such protein, Rab6, is associated with the Golgi apparatus and has a C-terminal CXC motif that is geranylgeranylated on both cysteines. We show here that farnesylation alone cannot substitute for geranylgeranylation in targeting Rab6 to the Golgi apparatus and that whereas Ras proteins that are farnesylated and palmitoylated are targeted to the plasma membrane, mutant Rab proteins that are both farnesylated and palmitoylated associate with the Golgi apparatus. Using chimeric Ras-Rab proteins, we find that there are sequences in the N-terminal 71 amino acids of Rab6 which are required for Golgi complex localization and show that these sequences comprise or include the effector domain. The C-terminal hypervariable domain is not essential for the Golgi complex targeting of Rab6 but is required to prevent prenylated and palmitoylated Rab6 from localizing to the plasma membrane. Functional analysis of these mutant Rab6 proteins in Saccharomyces cerevisiae shows that wild-type Rab6 and C-terminal mutant Rab6 proteins which localize to the Golgi apparatus in mammalian cells can complement the temperature-sensitive phenotype of ypt6 null mutants. Interestingly, therefore, the C-terminal hypervariable domain of Rab6 is not required for this protein to function in S. cerevisiae.
Beranger
F
F
INSERM Unité 248, Faculté de Médecine Lariboisière-St. Louis, Paris, France.
Paterson
H
H
Powers
S
S
de Gunzburg
J
J
Hancock
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004285:Dogs; D005455:Fluorescent Antibody Technique; D005816:Genetic Complementation Test; D006056:Golgi Apparatus / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D011993:Recombinant Fusion Proteins; D016283:Proto-Oncogene Proteins p21(ras)