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

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.

Four-stranded mini microtubules formed by Prosthecobacter BtubAB show dynamic instability

“…as was also reported elsewhere, recently (10). Adding more BtubC beyond the number of BtubAB heterodimers in the filaments did not significantly increase the amount of BtubC that spun down.…”

“…How related BtubAB filaments and microtubules are, and the consequences of binding of the third gene in the btub gene cluster (6,10), have remained unclear.…”

“…We suggest that after the likely horizontal gene transfer of the btubABC genes from a eukaryotic organism (7, 16) (or, alternatively, its evolution from a primitive tubulin precursor) (24), the size of the microtubules was reduced during subsequent evolution, possibly to fit better into the much smaller bacterial cells where they acquired a yet to be determined function. It will be especially interesting to see whether mini microtubules have a role in stalk formation in Prosthecobacter because they tend to be located there (8) and how regulation of filament dynamics by BtubC is used in the cellular context, especially because it was reported recently that BtubC might link mini microtubules to biological membranes (10). In the meantime, BtubABC will provide an exciting biochemical tool for the detailed atomistic investigation of filament dynamics, including treadmilling and dynamic instability, and will also provide an exciting building block for synthetic biology approaches to molecular machines.…”

Bacterial Tubulins A and B Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis

“…However, single BtubA/B filaments formed in vitro and observed using negative stain electron microscopy (EM) have been interpreted to consist of two parallel protofilaments (2,3), although some filament bundles have appeared to form a lumen (2). Observations with cryoelectron microscopy of in vitro assembled filaments are also consistent with helical polymers composed of 2 to 6 protofilaments (9). Based on results of pelleting assays (2,3) and mutagenesis studies (10), it has been inferred that BtubA/B protofilaments contain a polar, alternating linear arrangement of BtubA/B heterodimers.…”

“…1B) appear to be more tightly BtubA/B bundled than those observed in Prosthecobacter (8), but bundles of several filaments are visible in the cells, suggesting that the bundled form may be functionally relevant. The difference in the tightness of the bundle may be attributed to the protein Bklc of the btub operon, which is expressed in Prosthecobacter and has been shown to interact with both BtubA/B filaments and lipid membranes in vitro (9). Although in vitro, Bklc does not change the bundling of BtubA/B polymers (9), in vivo, looser bundling may occur as a consequence of association with the cell membrane.…”