Mgr2 functions as lateral gatekeeper for preprotein sorting in the mitochondrial inner membrane.

The majority of preproteins destined for mitochondria carry N-terminal presequences. The presequence translocase of the inner mitochondrial membrane (TIM23 complex) plays a central role in protein sorting. Preproteins are either translocated through the TIM23 complex into the matrix or are laterally released into the inner membrane. We report that the small hydrophobic protein Mgr2 controls the lateral release of preproteins. Mgr2 interacts with preproteins in transit through the TIM23 complex. Overexpression of Mgr2 delays preprotein release, whereas a lack of Mgr2 promotes preprotein sorting into the inner membrane. Preproteins with a defective inner membrane sorting signal are translocated into the matrix in wild-type mitochondria but are released into the inner membrane in Mgr2-deficient mitochondria. We conclude that Mgr2 functions as a lateral gatekeeper of the mitochondrial presequence translocase, providing quality control for the membrane sorting of preproteins.

Job contenders: roles of the ß-barrel assembly machinery and the translocation and assembly module in autotransporter secretion.

In Gram-negative bacteria, autotransporters secrete effector protein domains that are linked to virulence. Although they were once thought to be simple and autonomous secretion machines, mounting evidence reveals that multiple factors of the bacterial envelope are necessary for autotransporter assembly. Secretion across the outer membrane of their soluble effector "passenger domain" is promoted by the assembly of an outer membrane-spanning "ß-barrel domain". Both reactions require BamA, an essential component of the ß-barrel assembly machinery (BAM complex) that catalyzes the final reaction step by which outer membrane proteins are integrated into the lipid bilayer. A large amount of data generated in the last decade has shed key insights onto the mechanistic coordination of autotransporter ß-barrel domain assembly and passenger domain secretion. These results, together with the recently solved structures of the BAM complex, offer an unprecedented opportunity to discuss a detailed model of autotransporter assembly. Importantly, some autotransporters benefit from the presence of an additional machinery, the translocation and assembly module (TAM), a two-membrane spanning complex, which contains a BamA-homologous subunit. Although it remains unclear how the BAM complex and the TAM cooperate, it is evident that multiple preparatory steps are necessary for efficient autotransporter biogenesis.

The presequence pathway is involved in protein sorting to the mitochondrial outer membrane.

The mitochondrial outer membrane contains integral α-helical and ß-barrel proteins that are imported from the cytosol. The machineries importing ß-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane.

Mechanistic link between ß barrel assembly and the initiation of autotransporter secretion.

Autotransporters are bacterial virulence factors that contain an N-terminal extracellular ("passenger") domain and a C-terminal ß barrel ("ß") domain that anchors the protein to the outer membrane. The ß domain is required for passenger domain secretion, but its exact role in autotransporter biogenesis is unclear. Here we describe insights into the function of the ß domain that emerged from an analysis of mutations in the Escherichia coli O157:H7 autotransporter EspP. We found that the G1066A and G1081D mutations slightly distort the structure of the ß domain and delay the initiation of passenger domain translocation. Site-specific photocrosslinking experiments revealed that the mutations slow the insertion of the ß domain into the outer membrane, but do not delay the binding of the ß domain to the factor that mediates the insertion reaction (the Bam complex). Our results demonstrate that the ß domain does not simply target the passenger domain to the outer membrane, but promotes translocation when it reaches a specific stage of assembly. Furthermore, our results provide evidence that the Bam complex catalyzes the membrane integration of ß barrel proteins in a multistep process that can be perturbed by minor structural defects in client proteins.

Sequential and spatially restricted interactions of assembly factors with an autotransporter beta domain.

Autotransporters are bacterial virulence factors that consist of an N-terminal extracellular ("passenger") domain and a C-terminal ß barrel domain ("ß domain") that resides in the outer membrane. Here we used an in vivo site-specific photocrosslinking approach to gain insight into the mechanism by which the ß domain is integrated into the outer membrane and the relationship between ß domain assembly and passenger domain secretion. We found that periplasmic chaperones and specific components of the ß barrel assembly machinery (Bam) complex interact with the ß domain of the Escherichia coli O157:H7 autotransporter extracellular serine protease P (EspP) in a temporally and spatially regulated fashion. Although the chaperone Skp initially interacted with the entire ß domain, BamA, BamB, and BamD subsequently interacted with discrete ß domain regions. BamB and BamD remained bound to the ß domain longer than BamA and therefore appeared to function at a later stage of assembly. Interestingly, we obtained evidence that the completion of ß domain assembly is regulated by an intrinsic checkpoint mechanism that requires the completion of passenger domain secretion. In addition to leading to a detailed model of autotransporter biogenesis, our results suggest that the lipoprotein components of the Bam complex play a direct role in the membrane integration of ß barrel proteins.

Analysis of Transmembrane ß-Barrel Proteins by Native and Semi-native Polyacrylamide Gel Electrophoresis.

Membrane-embedded ß-barrel proteins are important regulators of the outer membrane permeability barrier of Gram-negative bacteria. ß-barrels are highly structured domains formed by a series of antiparallel ß-strands. Each ß-strand is locked in position by hydrogen bonds between its polypeptide backbone and those of the two neighbouring strands in the barrel structure. Some transmembrane ß-barrel proteins form larger homo- or hetero-multimeric complexes that accomplish specific functions. In this chapter, we describe native and semi-native polyacrylamide gel electrophoresis (PAGE) methods to characterize the organization of transmembrane ß-barrel proteins. We illustrate blue native (BN)-PAGE as an analytical method to assess the formation of protein complexes. Furthermore, we describe a heat-modifiability assay via semi-native PAGE as a rapid method to investigate the folding of transmembrane ß-barrels.

Secretion of a bacterial virulence factor is driven by the folding of a C-terminal segment.

Autotransporters are bacterial virulence factors consisting of an N-terminal "passenger domain" that is secreted in a C- to-N-terminal direction and a C-terminal "ß domain" that resides in the outer membrane (OM). Although passenger domain secretion does not appear to use ATP, the energy source for this reaction is unknown. Here, we show that efficient secretion of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP requires the stable folding of a C-terminal ≈17-kDa passenger domain segment. We found that mutations that perturb the folding of this segment do not affect its translocation across the OM but impair the secretion of the remainder of the passenger domain. Interestingly, an examination of kinetic folding mutants strongly suggested that the ≈17-kDa segment folds in the extracellular space. By mutagenizing the ≈17-kDa segment, we also fortuitously isolated a unique translocation intermediate. Analysis of this intermediate suggests that a heterooligomer that facilitates the membrane integration of OM proteins (the Bam complex) also promotes the surface exposure of the ≈17-kDa segment. Our results provide direct evidence that protein folding can drive translocation and help to clarify the mechanism of autotransporter secretion.

LptM promotes oxidative maturation of the lipopolysaccharide translocon by substrate binding mimicry.

Insertion of lipopolysaccharide (LPS) into the bacterial outer membrane (OM) is mediated by a druggable OM translocon consisting of a ß-barrel membrane protein, LptD, and a lipoprotein, LptE. The ß-barrel assembly machinery (BAM) assembles LptD together with LptE at the OM. In the enterobacterium Escherichia coli, formation of two native disulfide bonds in LptD controls translocon activation. Here we report the discovery of LptM (formerly YifL), a lipoprotein conserved in Enterobacteriaceae, that assembles together with LptD and LptE at the BAM complex. LptM stabilizes a conformation of LptD that can efficiently acquire native disulfide bonds, whereas its inactivation makes disulfide bond isomerization by DsbC become essential for viability. Our structural prediction and biochemical analyses indicate that LptM binds to sites in both LptD and LptE that are proposed to coordinate LPS insertion into the OM. These results suggest that, by mimicking LPS binding, LptM facilitates oxidative maturation of LptD, thereby activating the LPS translocon.

Interaction of an autotransporter passenger domain with BamA during its translocation across the bacterial outer membrane.

Autotransporters are a superfamily of virulence factors produced by Gram-negative bacteria consisting of a large N-terminal extracellular domain ("passenger domain") and a C-terminal beta barrel domain ("beta domain"). The mechanism by which the passenger domain is translocated across the outer membrane (OM) is unknown. Here we show that the insertion of a small linker into the passenger domain of the Escherichia coli O157:H7 autotransporter EspP effectively creates a translocation intermediate by transiently stalling translocation near the site of the insertion. Using a site-specific photocrosslinking approach, we found that residues adjacent to the stall point interact with BamA, a component of a heterooligomeric complex (Bam complex) that catalyzes OM protein assembly, and that residues closer to the EspP N terminus interact with the periplasmic chaperones SurA and Skp. The EspP-BamA interaction was short-lived and could be detected only when passenger domain translocation was stalled. These results support a model in which molecular chaperones prevent misfolding of the passenger domain before its secretion and the Bam complex catalyzes both the integration of the beta domain into the OM and the translocation of the passenger domain across the OM in a C- to N-terminal direction.

Lipoprotein DolP supports proper folding of BamA in the bacterial outer membrane promoting fitness upon envelope stress.

In Proteobacteria, integral outer membrane proteins (OMPs) are crucial for the maintenance of the envelope permeability barrier to some antibiotics and detergents. In Enterobacteria, envelope stress caused by unfolded OMPs activates the sigmaE (σE) transcriptional response. σE upregulates OMP biogenesis factors, including the ß-barrel assembly machinery (BAM) that catalyses OMP folding. Here we report that DolP (formerly YraP), a σE-upregulated and poorly understood outer membrane lipoprotein, is crucial for fitness in cells that undergo envelope stress. We demonstrate that DolP interacts with the BAM complex by associating with outer membrane-assembled BamA. We provide evidence that DolP is important for proper folding of BamA that overaccumulates in the outer membrane, thus supporting OMP biogenesis and envelope integrity. Notably, mid-cell recruitment of DolP had been linked to regulation of septal peptidoglycan remodelling by an unknown mechanism. We now reveal that, during envelope stress, DolP loses its association with the mid-cell, thereby suggesting a mechanistic link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.

Transmembrane Coordination of Preprotein Recognition and Motor Coupling by the Mitochondrial Presequence Receptor Tim50.

Mitochondrial preproteins contain amino-terminal presequences directing them to the presequence translocase of the mitochondrial inner membrane (TIM23 complex). Depending on additional downstream import signals, TIM23 either inserts preproteins into the inner membrane or translocates them into the matrix. Matrix import requires the coupling of the presequence translocase-associated motor (PAM) to TIM23. The molecular mechanisms coordinating preprotein recognition by TIM23 in the intermembrane space (IMS) with PAM activation in the matrix are unknown. Here we show that subsequent to presequence recognition in the IMS, the Tim50 matrix domain facilitates the recruitment of the coupling factor Pam17. Next, the IMS domain of Tim50 promotes PAM recruitment to TIM23. Finally, the Tim50 transmembrane segment stimulates the matrix-directed import-driving force exerted by PAM. We propose that recognition of preprotein segments in the IMS and transfer of signal information across the inner membrane by Tim50 determine import motor activation.

The Mgr2 subunit of the TIM23 complex regulates membrane insertion of marginal stop-transfer signals in the mitochondrial inner membrane.

The TIM23 complex mediates membrane insertion of presequence-containing mitochondrial proteins via a stop-transfer mechanism. Stop-transfer signals consist of hydrophobic transmembrane segments and flanking charges. Mgr2 functions as a lateral gatekeeper of the TIM23 complex. However, it remains elusive which features of stop-transfer signals are discriminated by Mgr2. To determine the effects of Mgr2 on the TIM23-mediated stop-transfer pathway, we measured membrane insertion of model transmembrane segments of varied hydrophobicity and flanking charges in Mgr2-deletion or -overexpression yeast strains. We found that upon deletion of Mgr2, the threshold hydrophobicity for membrane insertion, as well as the requirement for matrix-facing positive charges, is reduced. These results imply that the Mgr2-mediated gatekeeper function is important for controlling membrane sorting of marginal stop-transfer signals.

OxyR tightly regulates catalase expression in Neisseria meningitidis through both repression and activation mechanisms.

Mechanisms for coping with oxidative stress (OS) are crucial for the survival of pathogenic Neisseria spp. in the human host. In this study we investigate the mechanism by which OxyR finely regulates the catalase gene (kat) in Neisseria meningitidis. Detailed transcriptional analyses show that catalase is transcribed from a single promoter that is induced by H(2)O(2) in an OxyR-dependent manner and two key cysteine residues are essential for this. OxyR also represses the kat promoter: kat expression in the null mutant is at a constitutive intermediary level higher than uninduced, but lower than H(2)O(2)-induced levels in the wild type. Our data are consistent with a model in which OxyR binds to the kat promoter and exerts: (i) repression of transcription in the absence of OS signal and (ii) activation of the promoter in response to OS signal. This direct double-edged mechanism may ensure tight regulatory control of kat expression ensuring catalase is synthesized only when needed. In addition, our results provide an explanation for the altered OS resistance phenotypes seen in Neisseria mutant strains where, paradoxically, the oxyR mutants are more resistant than the wild type in oxidative killing assays.

Mitochondrial presequence import: Multiple regulatory knobs fine-tune mitochondrial biogenesis and homeostasis.

Mitochondria are pivotal organelles for cellular signaling and metabolism, and their dysfunction leads to severe cellular stress. About 60-70% of the mitochondrial proteome consists of preproteins synthesized in the cytosol with an amino-terminal cleavable presequence targeting signal. The TIM23 complex transports presequence signals towards the mitochondrial matrix. Ultimately, the mature protein segments are either transported into the matrix or sorted to the inner membrane. To ensure accurate preprotein import into distinct mitochondrial sub-compartments, the TIM23 machinery adopts specific functional conformations and interacts with different partner complexes. Regulatory subunits modulate the translocase dynamics, tailoring the import reaction to the incoming preprotein. The mitochondrial membrane potential and the ATP generated via oxidative phosphorylation are key energy sources in driving the presequence import pathway. Thus, mitochondrial dysfunctions have rapid repercussions on biogenesis. Cellular mechanisms exploit the presequence import pathway to monitor mitochondrial dysfunctions and mount transcriptional and proteostatic responses to restore functionality.

Bacterial machineries for the assembly of membrane-embedded ß-barrel proteins.

The outer membrane (OM) of Gram-negative bacteria is an essential organelle that protects cells from external aggressions and mediates the secretion of virulence factors. Efficient assembly of integral OM ß-barrel proteins (OMPs) is crucial for the correct functioning of the OM. Biogenesis of OMPs occurs in a stepwise manner that is finalized by the ß-barrel assembly machinery (BAM complex). Some OMPs further require the translocation and assembly module (TAM) for efficient and correct integration into the OM. Both the BAM complex and the TAM contain a protein of the Omp85 superfamily and distinct interacting factors. Their mechanism of action, however, remains largely elusive. We summarize and discuss recent structural and biochemical analyses that are helping to elucidate the molecular pathways of OMP assembly.

Site-Directed and Time-Resolved Photocrosslinking in Cells Metabolically Labeled with Radioisotopes.

To efficiently transport proteins into and across cellular membranes, specialized transport machineries engage in recognition events with different domains of their client proteins, forming sequential intermediate complexes. The short-lived nature of these interactions poses a big challenge in the identification of the key factors involved in transport reactions and their mechanism of action. Site-directed photocrosslinking is a powerful method for the detection and accurate mapping of interacting protein domains. This chapter describes a protocol that combines site-directed photocrosslinking to metabolic labeling of proteins and lipids as a method to characterize, with temporal and spatial resolution, the interactions of a secretory protein as it transverses the bacterial envelope.

Analysis of Mitochondrial Membrane Protein Complexes by Electron Cryo-tomography.

The visualization of membrane protein complexes in their natural membrane environment is a major goal in an emerging area of research termed structural cell biology. Such approaches provide important information on the spatial distribution of protein complexes in their resident cellular membrane systems and on the structural organization of multi-subunit membrane protein assemblies. We have developed a method to specifically label active membrane protein complexes in their native membrane environment with electron-dense nanoparticles coupled to an activating ligand, in order to visualize them by electron cryo-tomography. As an example, we describe here the depiction of preprotein import sites of mitochondria, formed by the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). Active import sites are selectively labeled via a biotinylated, quantum dot-coupled preprotein that is arrested in translocation across the outer and inner mitochondrial membranes. Additionally, a related method is described for direct labeling of mitochondrial outer membrane proteins that does not depend on binding of a ligand.

Two distinct membrane potential-dependent steps drive mitochondrial matrix protein translocation.

Two driving forces energize precursor translocation across the inner mitochondrial membrane. Although the membrane potential (Δψ) is considered to drive translocation of positively charged presequences through the TIM23 complex (presequence translocase), the activity of the Hsp70-powered import motor is crucial for the translocation of the mature protein portion into the matrix. In this study, we show that mitochondrial matrix proteins display surprisingly different dependencies on the Δψ. However, a precursor's hypersensitivity to a reduction of the Δψ is not linked to the respective presequence, but rather to the mature portion of the polypeptide chain. The presequence translocase constituent Pam17 is specifically recruited by the receptor Tim50 to promote the transport of hypersensitive precursors into the matrix. Our analyses show that two distinct Δψ-driven translocation steps energize precursor passage across the inner mitochondrial membrane. The Δψ- and Pam17-dependent import step identified in this study is positioned between the two known energy-dependent steps: Δψ-driven presequence translocation and adenosine triphosphate-driven import motor activity.

Protein Import by the Mitochondrial Presequence Translocase in the Absence of a Membrane Potential.

The highly organized mitochondrial inner membrane harbors enzymes that produce the bulk of cellular ATP via oxidative phosphorylation. The majority of inner membrane protein precursors are synthesized in the cytosol. Precursors with a cleavable presequence are imported by the presequence translocase (TIM23 complex), while other precursors containing internal targeting signals are imported by the carrier translocase (TIM22 complex). Both TIM23 and TIM22 are activated by the transmembrane electrochemical potential. Many small inner membrane proteins, however, do not resemble canonical TIM23 or TIM22 substrates and their mechanism of import is unknown. We report that subunit e of the F1Fo-ATP synthase, a small single-spanning inner membrane protein that is critical for inner membrane organization, is imported by TIM23 in a process that does not require activation by the membrane potential. Absence of positively charged residues at the matrix-facing amino-terminus of subunit e facilitates membrane potential-independent import. Instead, engineered positive charges establish a dependence of the import reaction on the electrochemical potential. Our results have two major implications. First, they reveal an unprecedented pathway of protein import into the mitochondrial inner membrane, which is mediated by TIM23. Second, they directly demonstrate the role of the membrane potential in driving the electrophoretic transport of positively charged protein segments across the inner membrane.

Visualizing active membrane protein complexes by electron cryotomography.

Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future.