As shown above, γ-BAR associates with membranes via palmitate lipid anchors attached to cysteine residues at its amino-terminal end. Membrane association might in turn influence binding to conventional kinesins. To investigate this possibility, we transfected Cos7 cells with FLAG-tagged γ-BAR wild type as well as the palmitoylation-defective mutant C4,5,9S and performed co-IPs using anti-FLAG tag antibodies. Then, we analyzed the samples for the presence of endogenous proteins associated with transfected γ-BAR by immunoblotting.
Binding of γ-BAR to conventional kinesins and AP-1 seems to be independent of its membrane association. When expressed in fibroblasts, the palmitoylation-defective mutant of γ-BAR bound to endogenous Klc2 and γ-adaptin to the same extent as the wild type protein (Figure 3-15, lane 2 and 3). None of the proteins was precipitated by the antibody using protein extracts from non-transfected cells (lane 1).
Results 67
Figure 3-15: Transfected γ-BAR FL and the C4,5,9S mutant bind Klc2 and γ-adaptin
Cos7 cells were transiently transfected with expression plasmids encoding FLAG-tagged γ-BAR or the palmitoylation-defective mutant (C4,5,9S) and Triton X-100 cell extracts (350 µg total protein) were used for co-immunoprecipitation using anti-FLAG tag antibodies immobilized on beads (α-FLAG IP). Endogenous proteins that bound to γ-BAR were analyzed by subjecting the samples to SDS-PAGE and immunoblotting using antibodies against FLAG-tag, Klc2 and γ-adaptin (AP1). Input corresponds to 30 µg (8%) of the cell lysates used for immunoprecipitation.
3.4.5 The carboxy-terminal domain of γ-BAR is required for kinesin binding
We further analyzed which part of γ-BAR is required for kinesin binding. To this aim, two γ-BAR truncation mutants fused to the FLAG-epitope tag were generated. The first mutant comprised amino acids 1 to 140, the second amino acids 140 to 302 of human γ- BAR. Constructs expressing either protein were transfected in Cos7 cells, cell extracts were prepared and used for co-IPs as before. Full length γ-BAR was expressed as a control.
As expected, full length γ-BAR bound to both Klc2 and γ-adaptin (AP-1), whereas the carboxy-terminal fragment of γ-BAR (aa 140-302) was able to bind to Klc2 only (Figure 3-16, lane 2 and 4). In contrast, the amino-terminal fragment (aa 1-140) associated only with γ-adaptin but not Klc2 (lane 3). We conclude, that the binding sites within γ-BAR for AP-1 and Klc2 are distinct.
Figure 3-16: AP-1 and Klc2 bind different parts of γ-BAR
Expression plasmids encoding FLAG-tagged γ-BAR full length (FL), aa 1-140 and aa 140-302 constructs were transfected into Cos7 cells. Triton X-100 extracted cell lysates (300 µg total protein) were incubated with anti-FLAG tag antibodies coupled to beads (α-FLAG IP). Bound proteins were analyzed by SDS- PAGE and immunoblotting using antibodies against γ-adaptin (AP-1), Klc2 and FLAG tag. Input corresponds to 25 µg (8%) of the cell lysates used for immunoprecipitation.
3.4.6 The carboxy-terminal part of γ-BAR harbors a tryptophan-based acidic motif important for kinesin binding
Recently, Calsyntenin-1, a neuronal transmembrane protein of the cadherin superfamily of adhesion molecules, was found to directly interact with the TPR motifs of kinesin light chain 1 (Klc1) via its cytoplasmic domain (Konecna et al., 2006). Calsyntenin-1 was found in a subset of vesicles aligned along microtubules in growth cones of primary cortical neurons and shows dynamic properties typical for kinesin-mediated transport. Thus, it might be a cargo-docking protein for kinesin-1-mediated vesicular transport. The binding of calsyntenin-1 to Klc1 is mediated by two conserved binding motifs, termed KBS1 and KBS2 (kinesin-binding segments 1 and 2). These binding motifs have the consensus sequence L/M-E/D-W-D-D-S. Mutation of the tryptophans to alanines in both motifs almost completely abolished the binding to Klc1, whereas single mutations in either of the motifs reduced the binding to about 30%. The carboxy-terminal part of γ-BAR, which binds to Klc2, contains two similar motifs: L-E-W-E-D-E (aa 208-213) and L-E-W-E-N-D (aa 258-263) with the first motif being more closely related to the
Results 69 defined consensus sequence. Both motifs are completely conserved between human, mouse and rat γ-BAR. We generated single point mutants of γ-BAR by mutating the tryptophans within either of the two motifs to alanines (γ-BAR W210A and γ-BAR W260A) as well as a double point mutant (γ-BAR W210/260A). GST-tagged γ-BAR Δ51 mutants were then used in pulldown assays from rat brain extracts to analyze their binding to conventional kinesin (Figure 3-17). We found that the first putative kinesin light chain binding motif of γ-BAR (aa 208-213) seems to be important for the association with kinesin, since a tryptophan to alanine exchange at position 210 completely abolished binding (lane 3). Mutation of the tryptophan residue within the second motif (aa 258-263) did not have a major effect on Klc binding, although the binding of kinesin to that mutant construct seemed to be diminished (lane 4). None of the mutations affected binding to AP-1.
Figure 3-17: Mutation of a single tryptophan to alanine in a putative kinesin light chain binding motif of γ-BAR abolishes binding to conventional kinesin
(A) Sequence comparison between the kinesin-binding segments (KBS) 1 and 2 of Calsyntenin-1 (Cst1)
and the two similar motifs found in γ-BAR. Residues shown to be important for kinesin light chain binding (Konecna et al., 2006) are color-coded.
(B) GST, GST-γ-BAR Δ51 and point mutants thereof (W210A, W260A and the double mutant W210/ 260A) were purified and 20 µg of each fusion protein were incubated with 1.6 mg rat brain extract. Samples were subjected to SDS-PAGE and immunoblotting using antibodies against kinesin heavy chain (HC), γ-adaptin (AP-1) and clathrin light chain as a control. The W210A mutation completely abolishes binding to conventional kinesin, whereas the interaction with AP-1 is unaffected. Std., standard corresponding to 50 µg rat brain extract (RBE).