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. 2011 Nov 16;30(22):4523-38.
doi: 10.1038/emboj.2011.326.

A kinesin-1 Binding Motif in Vaccinia Virus That Is Widespread Throughout the Human Genome

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Free PMC article

A kinesin-1 Binding Motif in Vaccinia Virus That Is Widespread Throughout the Human Genome

Mark P Dodding et al. EMBO J. .
Free PMC article

Abstract

Transport of cargoes by kinesin-1 is essential for many cellular processes. Nevertheless, the number of proteins known to recruit kinesin-1 via its cargo binding light chain (KLC) is still quite small. We also know relatively little about the molecular features that define kinesin-1 binding. We now show that a bipartite tryptophan-based kinesin-1 binding motif, originally identified in Calsyntenin is present in A36, a vaccinia integral membrane protein. This bipartite motif in A36 is required for kinesin-1-dependent transport of the virus to the cell periphery. Bioinformatic analysis reveals that related bipartite tryptophan-based motifs are present in over 450 human proteins. Using vaccinia as a surrogate cargo, we show that regions of proteins containing this motif can function to recruit KLC and promote virus transport in the absence of A36. These proteins interact with the kinesin light chain outside the context of infection and have distinct preferences for KLC1 and KLC2. Our observations demonstrate that KLC binding can be conferred by a common set of features that are found in a wide range of proteins associated with diverse cellular functions and human diseases.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
The WD/E motifs in A36 are required for efficient virus transport to the cell periphery. (A) Schematic representation of A36 highlighting its TM domain and positions of the WE and WD motifs at residues 64–65 and 97–98, respectively. (B) Representative immunofluorescence images showing actin tail formation (red) by the indicated A36–YFP WE/WD mutant viruses (green) at 8 h post-infection. Scale bar=10 μm. The graph shows quantification of the number of actin tails per cell for the indicated viruses. A P-value of <0.05 and <0.001 is indicated with * and ***, respectively, while error bars represent standard error of the mean from 50 cells in three independent experiments. (C) Representative immunofluorescence images showing the spread of the indicated recombinant A36–YdF–YFP viruses (green) from their perinuclear site of assembly to the cell periphery at 8 h post-infection compared with the ΔA36R virus. Scale bar=10 μm. The different viruses are co-labelled with anti-A27 (red), which detects both IMV and IEV, the latter of which appear yellow as they contain both A27 and A36–YdF–YFP. The graph shows quantification of viral spread to the cell periphery at 8 h post-infection based upon fluorescence intensity measurements of extracellular B5, an IEV-specific protein, detected with a monoclonal antibody in non-permeabilized cells. A P-value of <0.05 and <0.001 is indicated with * and ***, respectively. Error bars represent standard error of the mean from 50 cells in three independent experiments.
Figure 2
Figure 2
The A36 WD/E motifs are important for the cell-to-cell spread of the virus. (A) Representative fluorescence images of plaques produced by the WT, WE/AA, WD/AA and WDWE/AAAA viruses in the A36–YdF–YFP background at 48 h post-infection. Scale bar=100 μm. (B) Quantification of plaque sizes produced by the indicated viruses at 48 h post-infection. A P-value of <0.001 is indicated with *** and error bars represent s.e.m. from 30 plaques.
Figure 3
Figure 3
Both WD/E motifs contribute to KLC1/2 recruitment and viral spread. (A) Histograms and scatter plot showing the distribution of the speeds and run lengths of virus moving towards the cell periphery in cells infected with A36–YdF–YFP (black) as well as WE/AA (green) and WD/AA (red) viruses for 8 h. Inserts show the average values for both parameters and error bars represent s.e.m. for 65 virus particles in four independent experiments. (B) Immunofluorescence images showing RFP–KLC2 is not associated with the WD or WEWD viruses (yellow arrows) after they have fused with the plasma membrane at the cell periphery at 11 h post-infection. Pink arrows highlight association of RFP–KLC2 with DAPI-positive WT and WE viruses. Scale bar=10 μm. (C) Analysis of GST pull-down experiments showing the effect of mutating WD/WE motifs on the ability of the cytoplasmic domain of A36 to interact with HA–KLC1 (left panel), HA–KLC2 (middle panel) or endogenous kinesin-1 detected with anti-Kif5B antibody (right panel). Mutation of the WD motif weakens the association of A36 with KLC1/2 and kinesin-1, while mutation of both motifs abrogates all binding.
Figure 4
Figure 4
Bipartite tryptophan motifs of Calsyntenin can functionally replace A36. (A) Schematic representation of hybrid protein in which the region following the A36 TM domain (residue 32 onwards) is replaced with residues 879–971 of Calsyntenin. (B) Images showing the spread of the indicated recombinant A36–TM–CSTN1–GFP mutant viruses detected with anti-A27 (virus) from their perinuclear site of assembly to the cell periphery at 11 h post-infection. Scale bar=10 μm. (C) The graph shows quantification of extracellular B5 labelling for the indicated viruses. A P-value of <0.001 is indicated with *** and error bars represent s.e.m. from 50 cells in three independent experiments. (D) Images showing recruitment of RFP-tagged KLC1 and KLC2 to the WT A36–TM–CSTN1–GFP virus (GFP) co-labelled with DAPI to identify the viral genome. Scale bars=2 μm. (E) Representative fluorescence images of plaques produced by the A36–YdF–YFP, A36–TM–GFP and A36–TM–CSTN1–GFP viruses at 48 h post-infection. Scale bar=100 μm. (F) Quantification of plaque sizes produced by the indicated viruses at 48 h post-infection. A P-value of <0.001 is indicated with *** and error bars represent s.e.m. from 30 plaques.
Figure 5
Figure 5
Potential bipartite tryptophan KLC1/2-binding motifs are found in a wide range of proteins. (A) Sequence alignment showing the WD/E motifs in proteins previously shown to interact with KLC (bold text indicates experimental evidence). W–W indicates the number of residues separating the two tryptophans. A consensus based on these four proteins as well as conserved amino-acid substitutions is shown. A schematic representation of the set of patterns that was used to search the RefSeq database is shown. (B) Sequence alignment of bipartite tryptophan motifs in candidate KLC-binding proteins. SKIP, DYNC1I1 and HAP1 are known KLC-binding proteins. W–W indicates the number of residues between the two tryptophan residues highlighted in red. (C) Schematic representation of the location of bipartite tryptophan motifs in the proteins in (B) relative to other domains (from the Pfam database).
Figure 6
Figure 6
Multiple bipartite tryptophan motifs can rescue vaccinia transport. (A) Images showing the spread of the indicated recombinant A36–TM–SKIP–GFP viruses detected with anti-A27 (virus) from their perinuclear site of assembly to the cell periphery at 11 h post-infection. Scale bar=10 μm. (B) Quantification of viral spread to the cell periphery for the indicated recombinant A36–TM–SKIP–GFP viruses. A P-value of <0.001 is indicated with *** and error bars represent s.e.m. from 50 cells in three independent experiments. (C) Representative immunofluorescence showing the spread of A36–TM–ATF6–GFP and A36–TM–BSDC1–GFP viruses detected with anti-A27 (virus) from their perinuclear site of assembly to the cell periphery at 11 h post-infection. Scale bar=10 μm. (D) Quantification of viral spread to the cell periphery as detected by staining for extracellular B5 in the absence of permeabilization for the indicated recombinant viruses. A P-value of <0.001 is indicated with *** and error bars represent s.e.m. from 50 cells in three independent experiments. (E) Quantification of plaque sizes produced by the indicated recombinant viruses at 48 h post-infection. A P-value of <0.001 is indicated with *** and error bars represent s.e.m. from 30 plaques. (F) Quantification of transient rescue of viral spread to the cell periphery at 10 h post-infection. A P-value of <0.001 relative to the A36–TM control is indicated with *** and error bars represent s.e.m. from 50 cells in three independent experiments.
Figure 7
Figure 7
(A) Images showing recruitment of RFP-tagged KLC1 and KLC2 to the different recombinant virus particles. Purple arrows indicate virus particles (IEV) that show good recruitment of the indicated KLC isoform. The white arrows highlight weak recruitment of KLC2 by the A36–TM–BSDC1–GFP virus and yellow arrows indicate A36–TM–ATF6–GFP virus that cannot recruit KLC1. Scale bars=2 μm. (B) Western blot analysis of co-immunoprecipitation with the indicated antibodies reveals that FLAG–ATF6 associates with HA–KLC2 but not HA–KLC1 (left panel). In contrast, GFP–PARC interacts with HA–KLC1 but not HA–KLC2 (right panel). (C) Western blot analysis of co-immunoprecipitation experiments between GFP-tagged SKIP, BSDC1, FAM63B, LDLRAP1, PRKAG3, RASSF8 and RIC3 with either HA–KLC1 (left panel) or HA–KLC2 (right panel) reveals the candidate proteins can bind both isoforms or have distinct binding preferences for KLC1 or KLC2.

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