The effector domain of Rab6, plus a highly hydrophobic C terminus, is required for Golgi apparatus localization.

Abstract

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 Ras-related proteins are 21-to 25-kDa proteins which bind guanine nucleotides and have GTPase activity. On the basis of their sequence homology, they can be divided into three main subfamilies: Ras, Rho, and Rab (13). All operate as molecular switches by interacting with different sets of proteins in the inactive GDP-or active GTP-bound state (reviewed in reference 8). Ras-related proteins are involved in regulating a wide range of cellular processes, including cell proliferation, differentiation, intracellular trafficking (7, 24), membrane ruffling, and assembly of actin stress fibers (43,44). Although these proteins serve very diverse functions, their ability to associate with cell membranes is a fundamental requirement for biological activity (review in reference 20) such that mutations in Ras, Rho, and Rab proteins which block membrane localization also render these proteins biologically inactive (2,21,40,49,51).The mechanism of attachment of Ras proteins to the inner surface of the plasma membrane provides a model for the membrane targeting of other Ras-related proteins. The C terminus of Ras terminates in a CAAX motif (C = cysteine, A = aliphatic amino acid, X = any amino acid) which undergoes farnesylation of the cysteine residue (12, 28), proteolysis by an endopeptidase to remove the AAX amino acids (19, 23), and carboxyl-methyl esterification of the cysteine (16,23 (510) 222 9758. signal is a polylysine domain in K-Ras and cysteine palmitoylation sites in the H-and N-Ras proteins. This combination of CAAX motif plus a second signal is also sufficient to target cytosolic heterologous proteins, such as protein A (25) or p120 GTPase-activating protein (GAP) (34), to the plasma membrane.It is interesting to note that while Ras proteins are farnesylated, the majority of other Ras-related proteins are geranylgeranylated (reviewed in reference 29). Alkylation with C20 geranylgeranyl occurs on proteins with three different C-terminal motifs; CAAX motifs, where X is a leucine or a phenylalanine, are prenylated by geranylgeranyltransferase I (GGTase I) (11,41,47), and CC and CXC motifs, which are found at the C termini of Rab proteins, are prenylated by GGTase II, also known as Rab geranylgeranyltransferase (32,45,46). Following geranylgeranylation, CAAL/F motifs are AAX proteolysed and methylated. The CXC motif is geranylgeranylated on both cysteines and is also methylated (18,42). The stoichiometry of geranylgeranylation of the CC motif is less certain (20,36,38,50

Nucleotide induced conformation determines posttranslational isoprenylation of the ras related rab6 protein in insect cells

“…3A and B confirmed that the three different rab6 proteins are expressed in the Sf9 cells in similar amounts and in an native form as demonstrated by the GTPbinding and GTPase assays• To analyze the modification of the different rab6 proteins we used Triton X-114 extraction. It has previously been described that isoprenylated ras-like proteins are separated into the detergent-containing phase after lipid modification [17]. The results of the separation experiments are shown in Fig• 4A.…”

“…4A, lanes 1-4. The Triton X-114 extraction demonstrates that most of the rab6 wt and rab6 T27N molecules are isoprenylated in infected insect cells [17]. The extent of modification depends on the period between infection and cell harvest.…”

“…In addition parts of the rab proteins other than the C-terminal sequences are required for the rab's isoprenylation [32]. Recently Beranger et al reported that loop 3 and the hypervariable region of rab6 are structural requirements for isoprenylation [17]. In addition, certain point mutations in the effector domain of rablB inhibit lipidation [32].…”

“…transferase [17]. The same structures are also involved in targeting the rab6 protein to the Golgi apparatus [17].…”

“…The same structures are also involved in targeting the rab6 protein to the Golgi apparatus [17]. Studies on the related rab5 protein revealed that addition of geranylgeranyl residues depends on the nucleotide bound and thus on the protein conformation [18].…”