SKIP controls lysosome positioning using a composite kinesin-1 heavy and light chain-binding domain

Abstract

The molecular interplay between cargo recognition and regulation of the activity of the kinesin-1 microtubule motor is not well understood. Using the lysosome adaptor SKIP (also known as PLEKHM2) as model cargo, we show that the kinesin heavy chains (KHCs), in addition to the kinesin light chains (KLCs), can recognize tryptophan-acidic-binding determinants on the cargo when presented in the context of an extended KHC-interacting domain. Mutational separation of KHC and KLC binding shows that both interactions are important for SKIP–kinesin-1 interaction in vitro and that KHC binding is important for lysosome transport in vivo. However, in the absence of KLCs, SKIP can only bind to KHC when autoinhibition is relieved, suggesting that the KLCs gate access to the KHCs. We propose a model whereby tryptophan-acidic cargo is first recognized by KLCs, resulting in destabilization of KHC autoinhibition. This primary event then makes accessible a second SKIP-binding site on the KHC C-terminal tail that is adjacent to the autoinhibitory IAK region. Thus, cargo recognition and concurrent activation of kinesin-1 proceed in hierarchical stepwise fashion driven by a dynamic network of inter- and intra-molecular interactions.

Insights into Kinesin-1 Activation from the Crystal Structure of KLC2 Bound to JIP3

“…Binding of other factors to the KHC tail is then required to activate motility. This could be a cargo molecule ( Figures 8 B and 8C) ( Blasius et al., 2007 , Fu and Holzbaur, 2013 , Sun et al., 2011 , Watt et al., 2015 ), or, as suggested previously, the KLC acidic linker ( Figure 8 D) ( Yip et al., 2016 ), although recent work shows that robust activation of kinesin-1 by tryptophan-acidic cargoes requires cargo binding to the KHC tails ( Sanger et al., 2017 ). Detailed information on the structure of the intact kinesin-1 molecule and further mechanistic studies will be needed to investigate these issues further.…”

Structural basis for isoform-specific kinesin-1 recognition of Y-acidic cargo adaptors

“…Our previous work has demonstrated that binding of W-acidic motifs displaces an intramolecular interaction between the KLC TPR and the LFP-acidic region (Yip et al, 2016). Moreover, binding of W-acidic motifs or disruption of the LFP-acidic intramolecular interaction triggers global changes in the KLC conformation, promotes kinesin-1 activity and results in an additional binding site on KHCtail becoming accessible (Sanger et al, 2017;Yip et al, 2016). A key piece of data supporting this model is the observation that inclusion of the inhibitory LFP-containing sequence on KLC1/2 TPR substantially reduces its affinity for W-acidic peptides.…”

Structural basis for isoform-specific kinesin-1 recognition of Y-acidic cargo adaptors

“…Our previous work has demonstrated that binding of W-acidic motifs displaces an intramolecular interaction between the KLC TPR and the LFP-acidic region ( Yip et al, 2016 ). Moreover, binding of W-acidic motifs or disruption of the LFP-acidic intramolecular interaction triggers global changes in the KLC conformation, promotes kinesin-1 activity and results in an additional binding site on KHC-tail becoming accessible ( Sanger et al, 2017 ; Yip et al, 2016 ). A key piece of data supporting this model is the observation that inclusion of the inhibitory LFP-containing sequence on KLC1/2 TPR substantially reduces its affinity for W-acidic peptides.…”