Morais-Cabral, J. H. | Search bioRxiv

bioRxiv2023

c-di-AMP determines the hierarchical organization of bacterial RCK proteins

RCK domains or proteins are important regulatory components of cation and K+ channels and transporters both in eukaryotic and prokaryotic organisms. A lasting unanswered question about bacterial RCK proteins relates to the physiological role of the multiple, sometimes closely related, RCK genes encoded in the genome. We explored this question with the Ktr channels of Bacillus subtilis that include two genes encoding RCK proteins (KtrA and KtrC) and two genes encoding their membrane protein partners (KtrB and KtrD). Using a combination of in vivo characterization and in vitro functional analysis, we determined that the two RCK proteins are neither physiologically redundant or functionally equivalent. Instead, KtrC is the physiologically dominant RCK protein due to its ability to mediate K+ transport inactivation by c-di-AMP, a bacterial second messenger that is the master regulator of the K+ machinery in many species, while KtrA assembled channels are very insensitive to the dinucleotide. Moreover, KtrC and KtrA can form heteromeric assemblies that can control the Ktr channel activity and are sensitive to c-di-AMP inhibition. In parallel, we showed that conditions with a large number of Ktr channels assembled with KtrA, or with RCK proteins that do not mediate c-di-AMP inhibition, are toxic to the cell. Altogether, we have demonstrated that c-di-AMP regulation of Ktr channels goes beyond affecting transcription and functional activity, it also determines the hierarchical organization of bacterial RCK proteins.

microbiology↗

bioRxiv2021

JIP3 regulates bi-directional organelle transport in neurons through its interaction with dynein and kinesin-1

The conserved MAP kinase and motor scaffold JIP3 prevents excess lysosome accumulation in axons of vertebrates and invertebrates. Whether and how JIP3s interaction with dynein and kinesin-1 contributes to this critical organelle clearance function is unclear. Using purified recombinant human proteins, we show that dynein light intermediate chain (DLIC) binds to the N-terminal RH1 domain of JIP3, its paralog JIP4, and the lysosomal adaptor RILP. A point mutation in a hydrophobic pocket of the RH1 domain, previously shown to abrogate RILPL2 binding to myosin Va, abrogates the binding of JIP3/4 and RILP to DLIC without perturbing the interaction between the JIP3 RH1 domain and kinesin heavy chain. Characterization of this separation-of-function mutation in Caenorhabditis elegans shows that JIP3-bound dynein is required for organelle clearance in the anterior process of touch receptor neurons. Unlike JIP3 null mutants, JIP3 that cannot bind DLIC causes prominent accumulation of endo-lysosomal organelles at the neurite tip, which is rescued by a disease-associated point mutation in JIP3s leucine zipper that abrogates kinesin light chain binding. These results highlight that RH1 domains are interaction hubs for cytoskeletal motors and suggest that JIP3-bound dynein and kinesin-1 participate in bi-directional organelle transport.

cell biology↗