Bacterial kinesin light chain (Bklc) links the Btub cytoskeleton to membranes

Lurlène Akendengue,Martín Graña,Alexandre Chenal,Alexis Voegele,Sergio Marco,Bertrand Raynal,Anne Marie Wehenkel,Carsten Janke,Sylvain Trépout

doi:10.1038/srep45668

Abstract

Bacterial kinesin light chain is a TPR domain-containing protein encoded by the bklc gene, which co-localizes with the bacterial tubulin (btub) genes in a conserved operon in Prosthecobacter. Btub heterodimers show high structural homology with eukaryotic tubulin and assemble into head-to-tail protofilaments. Intriguingly, Bklc is homologous to the light chain of the microtubule motor kinesin and could thus represent an additional eukaryotic-like cytoskeletal element in bacteria. Using biochemical characterization as well as cryo-electron tomography we show here that Bklc interacts specifically with Btub protofilaments, as well as lipid vesicles and could thus play a role in anchoring the Btub filaments to the membrane protrusions in Prosthecobacter where they specifically localize in vivo. This work sheds new light into possible ways in which the microtubule cytoskeleton may have evolved linking precursors of microtubules to the membrane via the kinesin moiety that in today’s eukaryotic cytoskeleton links vesicle-packaged cargo to microtubules.

Highlights

Tubulin is one of the major components of the eukaryotic cytoskeleton and assembles as an obligate heterodimer into protofilaments that in turn form microtubules, which are generally made up of 13 protofilaments
In order to understand whether btubAB and bklc form a functional operon we set out to characterize Bklc with respect to the bacterial tubulins
Bklc was found to bind to lipid vesicles, and cryo-electron tomography allowed us to visualize a ternary complex between bacterial tubulins (Btubs) filaments, Bklc and lipids, strongly suggesting a role in the membrane anchorage of the Btub filaments within the prosthecae

Summary

Results and Discussion

For the same amount of protein, the signal from the Btubs on the Coomassie stained gel was about twice that of Bklc Relating these values to the S2 fraction of the sedimentation assay, the stoichiometry estimated corresponds to about 1 Bklc monomer per BtubA/B heterodimer. The presence of an N-terminal amphipathic helix as well as previous localization studies of Btubs within the base of the prosthecae and near the plasma membrane of P. vanneervenii[6] suggested that Bklc could bind to membranes and play a functional role in membrane tethering of the Btub filaments. We confirmed the Bklc-SUV interaction using cryo-EM where Ni-NTA nano-gold beads that label the C-terminal His-tag of Bklc were co-localized with lipid vesicles (Fig. 4B). Alpha-solenoid based architectures are evolutionary conserved modules involved in membrane trafficking and shaping[20] and it is thought that membrane-curving protein modules were required to evolve the endomembrane system[21]

Materials and Methods

Author Contributions

Additional Information