Kinesin-1 is a motor protein that is made up of two kinesin heavy chains (KHCs) – which are responsible for the motor activity of the complex, and two kinesin light chains (KLCs) – which bind cargo in many cases. This complex moves along microtubules to transport components, such as vesicles, mitochondria, and even viruses in infected cells. Much is known about the structure and biomechanical properties of kinesin-1, but less is known about its protein interactions. Previous studies have suggested that two tryptophan-based motifs (WE/WD) are important for kinesin-1 binding (Aoyama et al. 2009; Morgan et al., 2010). A paper by Dodding et al. (2011) has now taken this work further, showing that these motifs are not only found in a component of the vaccinia virus (and are important for its kinesin-1 binding and transport), but they are also found in hundreds of human proteins, including FLCN.
The authors noted that the WE/WD motif was present in the A36 protein of the vaccinia virus, a protein which is known to be involved in the transport of viruses to the cell surface. In order to study the function of the motif, mutated versions of the virus were generated. From this it could be seen that mutating the WE/WD motif led to a significant reduction in viruses reaching the host HeLa cell membrane (as measured by two independent immunofluorescence experiments). This result was mirrored in a viral plaque assay using BSC1 cells, which showed that viral spread was significantly reduced after mutation of the WE/WD residues. Upon further investigation, an association between the mutated motif and KLC1 and KLC2 was no longer detected using both immunofluorescence and pull-down assays. These results together suggest that the double tryptophan motif is involved in both kinesin-1 recruitment and transport.
The RefSeq database of expressed human proteins was then searched for similar motifs and 460 candidate proteins were identified, including FLCN and DENND1B. This is of particular interest as a recent paper by Nookala et al. (2012) has postulated that FLCN and DENND1B are structurally and functionally related to one another. Furthermore, experiments in which the motif-containing region of A36 was replaced with that from a number of other candidate proteins showed that these regions were functionally equivalent in terms of both viral transport and spread. Immunofluorescence and pull-down assays also demonstrated that a number of these proteins could bind KLC1 and KLC2 to differing extents.
Using the assays described in this paper, it would be interesting to see if the bipartite tryptophan motif in FLCN can contribute to KLC1/KLC2 binding and intracellular transport. These results would add to the work conducted by Nookala et al. (2012), which suggested that FLCN could be involved in membrane trafficking and cytokinesis.
- Aoyama T, Hata S, Nakao T, Tanigawa Y, Oka C, & Kawaichi M (2009). Cayman ataxia protein caytaxin is transported by kinesin along neurites through binding to kinesin light chains. Journal of cell science, 122 (Pt 22), 4177-85 PMID: 19861499
- Dodding MP, Mitter R, Humphries AC, & Way M (2011). A kinesin-1 binding motif in vaccinia virus that is widespread throughout the human genome. The EMBO journal, 30 (22), 4523-38 PMID: 21915095
- Morgan GW, Hollinshead M, Ferguson BJ, Murphy BJ, Carpentier DC, & Smith GL (2010). Vaccinia protein F12 has structural similarity to kinesin light chain and contains a motor binding motif required for virion export. PLoS pathogens, 6 (2) PMID: 20195521
- Nookala RK, Langemeyer L, Pacitto A, Ochoa-Montano B, Donaldson JC, Blaszczyk BK, Chirgadze DY, Barr FA, Bazan JF, Blundell TL (2012). Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer Open Biol. DOI: 10.1098/rsob.120071