It is now clear that transport on microtubules by dynein and kinesin family motors has an important if not critical role in the replication and spread of many different viruses. Understanding how viruses hijack dynein and kinesin motors using a limited repertoire of proteins offers a great opportunity to determine the molecular basis of motor recruitment. In this review, we discuss the interactions of dynein and kinesin‐1 with adenovirus, the α herpes viruses: herpes simplex virus (HSV1) and pseudorabies virus (PrV), human immunodeficiency virus type 1 (HIV‐1) and vaccinia virus. We highlight where the molecular links to these opposite polarity motors have been defined and discuss the difficulties associated with identifying viral binding partners where the basis of motor recruitment remains to be established. Ultimately, studying microtubule‐based motility of viruses promises to answer fundamental questions as to how the activity and recruitment of the dynein and kinesin‐1 motors are coordinated and regulated during bi‐directional transport.

Kinesin-mediated cargo transport is required for many cellular functions and plays a key role in pathological processes. Structural information on how kinesins recognize their cargoes is required for a molecular understanding of this fundamental and ubiquitous process. Here we present the crystal structure of the tetratricopeptide repeat of kinesin light chain 2 in complex with a cargo peptide harboring a 'tryptophan-acidic' motif derived from SKIP, a critical host determinant in Salmonella pathogenesis and a regulator of lysosomal positioning. Structural data together with biophysical, biochemical and cellular assays allow us to propose a framework for intracellular transport based on the binding by kinesin-1 of W-acidic cargo motifs through a combination of electrostatic interactions and sequence-specific elements, providing direct molecular evidence of the mechanisms for kinesin-1:cargo recognition.The plus-end directed motor, kinesin-1 plays a critical role in the intracellular transport of diverse protein, ribonuclear protein complexes and membrane compartments on microtubules (1). Its functions are also usurped by bacteria and viruses to aid in their replication (2, 3). Kinesin-1 can perform this diverse range of functions by virtue of its ability to interact with many different cargo proteins (4). Diversity of cargo recognition is accomplished largely through the kinesin light chains (KLC) which harbour a tetratricopeptide repeat (TPR) domain, a versatile protein interaction platform (5, 6). The KLC TPR domain can recognise short peptide stretches within relatively disordered regions of its targets. These peptides are characterized by a tryptophan residue flanked by acidic residues (e.g. EWD) and are found in a growing list of KLC binding proteins (7-16). Although W-acidic motifs often occur in pairs, single motifs are also functional and can support microtubule-based transport even when explanted from their host protein (7,12,17). We set out to solve the structure of a KLC TPR domain bound to a cargo W-acidic motif. We focused our attention on the SKIP cargo for its importance in Salmonella pathogenesis.SKIP contains a pair of W-acidic motifs centred at amino acid positions 207-208 (WD) and 236-237 (WE) that fall within the N-terminal kinesin-1 binding region (residues 1-310)(3, 7, 13) (Fig. 1A). To assess the relative importance of the SKIP W-acidic motifs for KLC binding we co-transfected HeLa cells with wild-type and WD/WE mutant constructs expressing GFP-SKIP(1-310) and HA-KLC2 (Fig. 1B). Disruption of the WD motif significantly reduced GFP-SKIP interaction with HA-KLC2 whereas abrogation of the WE motif had no obvious effects. A double mutant with both motifs disrupted displayed HA-KLC2 binding similar to the single WD mutant. Thus the WE motif has a very low affinity for KLC2. Indeed, a ten amino acid long peptide centered on the WD motif (SKIP WD , Figure 1A) bound to KLC2 TPR with a K D of 24 μM whereas the affinity of the equivalent SKIP WE peptide was above 110 μM (Fig. 1C). The presenc...

The light chains (KLCs) of the microtubule motor kinesin-1 bind cargoes and regulate its activity. Through their tetratricopeptide repeat domain (KLC TPR ), they can recognize short linear peptide motifs found in many cargo proteins characterized by a central tryptophan flanked by aspartic/glutamic acid residues (W-acidic). Using a fluorescence resonance energy transfer biosensor in combination with X-ray crystallographic, biochemical, and biophysical approaches, we describe how an intramolecular interaction between the KLC2 TPR domain and a conserved peptide motif within an unstructured region of the molecule, partly occludes the W-acidic binding site on the TPR domain. Cargo binding displaces this interaction, effecting a global conformational change in KLCs resulting in a more extended conformation. Thus, like the motor-bearing kinesin heavy chains, KLCs exist in a dynamic conformational state that is regulated by self-interaction and cargo binding. We propose a model by which, via this molecular switch, W-acidic cargo binding regulates the activity of the holoenzyme.kinesin | KLC | TPR domain | microtubule motor | cytoskeleton

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.

The spatial distribution of lysosomes is important for their function and is, in part, controlled by cellular nutrient status. Here, we show that the lysosome associated Birt–Hoge–Dubé (BHD) syndrome renal tumour suppressor folliculin (FLCN) regulates this process. FLCN promotes the peri‐nuclear clustering of lysosomes following serum and amino acid withdrawal and is supported by the predominantly Golgi‐associated small GTPase Rab34. Rab34‐positive peri‐nuclear membranes contact lysosomes and cause a reduction in lysosome motility and knockdown of FLCN inhibits Rab34‐induced peri‐nuclear lysosome clustering. FLCN interacts directly via its C‐terminal DENN domain with the Rab34 effector RILP. Using purified recombinant proteins, we show that the FLCN‐DENN domain does not act as a GEF for Rab34, but rather, loads active Rab34 onto RILP. We propose a model whereby starvation‐induced FLCN association with lysosomes drives the formation of contact sites between lysosomes and Rab34‐positive peri‐nuclear membranes that restrict lysosome motility and thus promote their retention in this region of the cell.