Proteins destined for the mitochondrial matrix are targeted to the inner membrane Tim17/23 translocon by their presequences. Inward movement is driven by the matrix-localized, Hsp70-based motor. The ...scaffold Tim44, interacting with the matrix face of the translocon, recruits other motor subunits and binds incoming presequence. The basis of these interactions and their functional relationships remains unclear. Using site-specific in vivo crosslinking and genetic approaches in
, we found that both domains of Tim44 interact with the major matrix-exposed loop of Tim23, with the C-terminal domain (CTD) binding Tim17 as well. Results of in vitro experiments showed that the N-terminal domain (NTD) is intrinsically disordered and binds presequence near a region important for interaction with Hsp70 and Tim23. Our data suggest a model in which the CTD serves primarily to anchor Tim44 to the translocon, whereas the NTD is a dynamic arm, interacting with multiple components to drive efficient translocation.
ADP-ribosylation of proteins can profoundly impact their function and serves as an effective mechanism by which bacterial toxins impair eukaryotic cell processes. Here, we report the discovery that ...bacteria also employ ADP-ribosylating toxins against each other during interspecies competition. We demonstrate that one such toxin from Serratia proteamaculans interrupts the division of competing cells by modifying the essential bacterial tubulin-like protein, FtsZ, adjacent to its protomer interface, blocking its capacity to polymerize. The structure of the toxin in complex with its immunity determinant revealed two distinct modes of inhibition: active site occlusion and enzymatic removal of ADP-ribose modifications. We show that each is sufficient to support toxin immunity; however, the latter additionally provides unprecedented broad protection against non-cognate ADP-ribosylating effectors. Our findings reveal how an interbacterial arms race has produced a unique solution for safeguarding the integrity of bacterial cell division machinery against inactivating post-translational modifications.
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•Protein-targeting ADP-ribosyltransferases (ARTs) act as interbacterial toxins•Immunity proteins protect from ARTs by toxin binding and ADP-ribose (ADPr) cleavage•A type VI secretion effector from Serratia acts via ADPr addition to FtsZ•FtsZ-ADPr blocks polymerization and cell division, leading to cell death
A widely conserved toxin for interbacterial competition targets proteins for ADP-ribosylation, while antitoxins fight back using dual mechanisms of active site occlusion and enzymatic reversal of the modification, conferring broad immunity to diverse toxins.
Abstract
Many AAA+ (ATPases associated with diverse cellular activities) proteins function as protein or DNA remodelers by threading the substrate through the central pore of their hexameric ...assemblies. In this ATP-dependent translocating state, the substrate is gripped by the pore loops of the ATPase domains arranged in a universal right-handed spiral staircase organization. However, the process by which a AAA+ protein is activated to adopt this substrate-pore-loop arrangement remains unknown. We show here, using cryo-electron microscopy (cryo-EM), that the activation process of the Lon AAA+ protease may involve a pentameric assembly and a substrate-dependent incorporation of the sixth protomer to form the substrate-pore-loop contacts seen in the translocating state. Based on the structural results, we design truncated monomeric mutants that inhibit Lon activity by binding to the native pentamer and demonstrated that expressing these monomeric mutants in
Escherichia coli
cells containing functional Lon elicits specific phenotypes associated with
lon
deficiency, including the inhibition of persister cell formation. These findings uncover a substrate-dependent assembly process for the activation of a AAA+ protein and demonstrate a targeted approach to selectively inhibit its function within cells.
Bacterial survival is fraught with antagonism, including that deriving from viruses and competing bacterial cells. It is now appreciated that bacteria mount complex antiviral responses; however, ...whether a coordinated defense against bacterial threats is undertaken is not well understood. Previously, we showed that
possess a danger-sensing pathway that is a critical fitness determinant during competition against other bacteria. Here, we conducted genome-wide screens in
that reveal three conserved and widespread interbacterial antagonism resistance clusters (
). We find that although
are coordinately activated by the Gac/Rsm danger-sensing system, they function independently and provide idiosyncratic defense capabilities, distinguishing them from general stress response pathways. Our findings demonstrate that Arc3 family proteins provide specific protection against phospholipase toxins by preventing the accumulation of lysophospholipids in a manner distinct from previously characterized membrane repair systems. These findings liken the response of
to bacterial threats to that of eukaryotic innate immunity, wherein threat detection leads to the activation of specialized defense systems.
Developing programmable bacterial cell-cell adhesion is of significant interest due to its versatile applications. Current methods that rely on presenting cell adhesion molecules (CAMs) on bacterial ...surfaces are limited by the lack of a generalizable strategy to identify such molecules targeting bacterial membrane proteins in their natural states. Here, we introduce a whole-cell screening platform designed to discover CAMs targeting bacterial membrane proteins within a synthetic bacteria-displayed nanobody library. Leveraging the potency of the bacterial type IV secretion system—a contact-dependent DNA delivery nanomachine—we have established a positive feedback mechanism to selectively enrich for bacteria displaying nanobodies that target antigen-expressing cells. Our platform successfully identified functional CAMs capable of recognizing three distinct outer membrane proteins (TraN, OmpA, OmpC), demonstrating its efficacy in CAM discovery. This approach holds promise for engineering bacterial cell-cell adhesion, such as directing the antibacterial activity of programmed inhibitor cells toward target bacteria in mixed populations.Cell adhesion molecules (CAMs) are key for programming bacterial cell-cell adhesion. By leveraging the transfer of selectable marker genes between bacterial cells, the authors present a method for discovering synthetic CAMs that target naturally occurring bacterial surface components.
Selective and targeted removal of individual species or strains of bacteria from complex communities can be desirable over traditional, broadly acting antibacterials in several contexts. However, ...generalizable strategies that accomplish this with high specificity have been slow to emerge. Here we develop programmed inhibitor cells (PICs) that direct the potent antibacterial activity of the type VI secretion system (T6SS) against specified target cells. The PICs express surface-displayed nanobodies that mediate antigen-specific cell–cell adhesion to effectively overcome the barrier to T6SS activity in fluid conditions. We demonstrate the capacity of PICs to efficiently deplete low-abundance target bacteria without significant collateral damage to complex microbial communities. The only known requirements for PIC targeting are a Gram-negative cell envelope and a unique cell surface antigen; therefore, this approach should be generalizable to a wide array of bacteria and find application in medical, research, and environmental settings.
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•Programmed inhibitor cells (PICs) recognize target bacteria via surface nanobodies•PICs selectively and potently deplete low-abundance targets via toxin delivery•PICs deplete target cells from complex communities with minimal off-target effects•Resistance to PICs recognizing an essential surface structure is slow to emerge
Few tools exist for the selective depletion of specific bacteria from complex mixtures, such as the gut microbiome. Ting et al. demonstrate that nanobody-based cell–cell adhesion enables efficient depletion of bacteria of interest from a complex community via the targeted delivery of toxic proteins by a programmable inhibitor cell.
Translocation of proteins from the cytosol across the mitochondrial inner membrane is driven by action of the matrix-localized multi-subunit import motor, which is associated with the TIM23 ...translocon. The architecture of the import apparatus is not well understood. Here, we report results of site-specific in vivo photocross-linking along with genetic and coimmunoprecipitation analyses dissecting interactions between import motor subunits and the translocon. The translocon is composed of the two integral membrane proteins Tim23 and Tim17, each containing four membrane-spanning segments. We found that Tim23 having a photoactivatable cross-linker in the matrix exposed loop between transmembrane domains 1 and 2 (loop 1) cross-linked to Tim44.
Alterations in this loop destabilized interaction of Tim44 with the translocon. Analogously, Tim17 having a photoactivatable cross-linker in the matrix exposed loop between transmembrane segments 1 and 2 (loop 1) cross-linked to Pam17. Alterations in this loop caused destabilization of the interaction of Pam17 with the translocon. Substitution of individual photoactivatable residues in Tim44 and Pam17 in regions we previously identified as important for translocon association resulted in cross-linking to Tim23 and Tim17, respectively. Our results are consistent with a model in which motor association is achieved via interaction of Tim23 with Tim44, which serves as a scaffold for association of other motor components, and of Tim17 with Pam17. As both Tim44 and Pam17 have been implicated as regulatory subunits of the motor, this positioning is conducive for responding to conformational changes in the translocon upon a translocating polypeptide entering the channel.
Abstract only
Because the vast majority of mitochondrial proteins are synthesized in the cytosol, efficient protein import is essential for mitochondrial function and cell viability. The ...mitochondrial protein import motor, which is tethered to the matrix face of the inner membrane, plays a critical role. This motor translocates preproteins from the cytosol, across both mitochondrial membranes, into the mitochondrial matrix.
This biological machine is driven by the molecular chaperone mtHsp70, which binds translocating preproteins entering the matrix in an ATP‐dependent manner. Both mtHsp70 and its co‐chaperone Pam18, which performs the critical function of stimulating Hsp70's ATPase activity, are tethered to the translocon by Tim44. While mtHsp70 directly interacts with Tim44, evidence suggests that Pam18 is tethered via Pam16, with which it forms a heterodimer. However, how Pam16 interacts with Tim44 is not well understood.
This study aims to determine how Pam16 interacts with Tim44 and the functional consequences of the interaction. Our previous genetic and co‐immunoprecipitation analyses implicated distinct regions in Pam16 and Tim44, both in their N‐termini, that may be the sites of interaction. Alterations in these regions not only caused Pam16 to dissociate from the translocon, but also led to temperature‐sensitive growth defects, demonstrating the importance of this interaction. We now report site‐specific crosslinking demonstrating that Pam16 and Tim44 are in close proximity to each other. When a photoactivatable crosslinker was incorporated at several positions in these regions, Pam16 could be crosslinked to Tim44, or vice versa. Altogether, these results suggest that Pam16 directly interacts with Tim44 via these regions.
In addition to crosslinking to Tim44, we found that the extreme N terminus of Pam16 also crosslinked to Tim50, an essential translocon subunit that facilitates movement of the preprotein from the translocon of the outer membrane to that of the inner membrane. Tim50 is a transmembrane protein with an essential intermembrane space domain and a matrix‐exposed region whose function is unknown. Taken together, these results support the idea of Pam16 as an essential tether in the import motor, mediating complex interactions with subunits that are necessary for mitochondrial protein import.
As nearly all of the proteins residing in the mitochondria are encoded by nuclear DNA and synthesized in the cytoplasm, mitochondrial biogenesis and function require efficient protein import ...mechanisms. Presequence-containing proteins destined for the mitochondrial matrix initially enter mitochondria via the TOM complex of the outer membrane and are translocated across the inner membrane into the matrix through the TIM23 complex. From here, they are completely imported into the matrix with the help of a matrix-localized Hsp70-based import motor. The association of the import motor with the TIM23 complex is key to efficient protein translocation. However, the organization of the import machinery and the regulatory mechanisms remain elusive. In this thesis, I report the results of experiments that better define the interactions within the TIM23-Motor complex, aiming to gain a deeper mechanistic understanding of the import machinery involved in mitochondrial protein translocation.The TIM23 complex is composed of two integral proteins, Tim23 and Tim17, each exposing a 24 amino acid loop to the matrix. Using in vivo site-specific crosslinking, along with genetic analyses, I found that motor association is achieved via interaction of the loop of Tim23 with Tim44, and the loop of Tim17 with Pam17. As both Tim44 and Pam17 have been implicated as regulatory subunits of the motor, this positioning is conducive for responding to conformational changes in the translocon upon a translocating polypeptide entering the channel.I further focused on Tim44, an essential two-domain motor subunit that plays a central role in recruiting and “organizing” the motor at the translocon. Using the same site-specific crosslinking approach, I showed that both domains of Tim44 interact with the 24 amino acid loop of Tim23, with the C-terminal domain binding Tim17 as well. Additionally, the N-terminal domain, which I found to be intrinsically disordered, interacts with presequence. Together, these results allow presentation of a more comprehensive model in which Tim44 not only serves as the molecular platform to tether the motor subunits to the tranlocon, but also uses its “dynamic arm” for interacting with multiple components to drive efficient translocation.
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