The Sec61 complex is the major protein translocation channel of the endoplasmic reticulum (ER), where it plays a central role in the biogenesis of membrane and secretory proteins. Whilst Sec61-mediated protein translocation is typically coupled to polypeptide synthesis, suggestive of significant complexity, an obvious characteristic of this core translocation machinery is its surprising simplicity. Over thirty years after its initial discovery, we now understand that the Sec61 complex is in fact the central piece of an elaborate jigsaw puzzle, which can be partly solved using new research findings. We propose that the Sec61 complex acts as a dynamic hub for co-translational protein translocation at the ER, proactively recruiting a range of accessory complexes that enhance and regulate its function in response to different protein clients. It is now clear that the Sec61 complex does not have a monopoly on co-translational insertion, with some transmembrane proteins preferentially utilising the ER membrane complex instead. We also have a better understanding of post-insertion events, where at least one membrane-embedded chaperone complex can capture the newly inserted transmembrane domains of multi-span proteins and co-ordinate their assembly into a native structure. Having discovered this array of Sec61-associated components and competitors, our next challenge is to understand how they act together in order to expand the range and complexity of the membrane proteins that can be synthesised at the ER. Furthermore, this diversity of components and pathways may open up new opportunities for targeted therapeutic interventions designed to selectively modulate protein biogenesis at the ER.

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Membrane protein biogenesis at the ER: the highways and byways.

Molecular chaperones are central to cellular protein homeostasis. Dynamic disorder is a key feature of the complexes of molecular chaperones and their client proteins, and it facilitates the client release towards a folded state or the handover to downstream components. The dynamic nature also implies that a given chaperone can interact with many different client proteins, based on physico-chemical sequence properties rather than on structural complementarity of their (folded) 3D structure. Yet, the balance between this promiscuity and some degree of client specificity is poorly understood. Here, we review recent atomic-level descriptions of chaperones with client proteins, including chaperones in complex with intrinsically disordered proteins, with membrane-protein precursors, or partially folded client proteins. We focus hereby on chaperone-client interactions that are independent of ATP. The picture emerging from these studies highlights the importance of dynamics in these complexes, whereby several interaction types, not only hydrophobic ones, contribute to the complex formation. We discuss these features of chaperone-client complexes and possible factors that may contribute to this balance of promiscuity and specificity.

How do Chaperones Bind (Partly) Unfolded Client Proteins

Recently, we presented a whole-cell kinetic model of the genetically minimal bacterium JCVI-syn3A that described the coupled metabolic and genetic information processes and predicted behaviors emerging from the interactions among these networks. JCVI-syn3A is a genetically reduced bacterial cell that has the fewest number and smallest fraction of genes of unclear function, with approximately 90 of its 452 protein-coding genes (that is less than 20%) unannotated. Further characterization of unclear JCVI-syn3A genes strengthens the robustness and predictive power of cell modeling efforts and can lead to a deeper understanding of biophysical processes and pathways at the cell scale. Here, we apply computational analyses to elucidate the functions of the products of several essential but previously uncharacterized genes involved in integral cellular processes, particularly those directly affecting cell growth, division, and morphology. We also suggest directed wet-lab experiments informed by our analyses to further understand these “missing puzzle pieces” that are an essential part of the mosaic of biological interactions present in JCVI-syn3A. Our workflow leverages evolutionary sequence analysis, protein structure prediction, interactomics, and genome architecture to determine upgraded annotations. Additionally, we apply the structure prediction analysis component of our work to all 452 protein coding genes in JCVI-syn3A to expedite future functional annotation studies as well as the inverse mapping of the cell state to more physical models requiring all-atom or coarse-grained representations for all JCVI-syn3A proteins.

Toward the Complete Functional Characterization of a Minimal Bacterial Proteome

In 1971, Blobel proposed the first statement of the Signal Hypothesis which suggested that proteins have amino-terminal sequences that dictate their export and localization in the cell. A cytosolic binding factor was predicted, and later the protein conducting channel was discovered that was proposed in 1975 to align with the large ribosomal tunnel. The 1975 Signal Hypothesis also predicted that proteins targeted to different intracellular membranes would possess distinct signals and integral membrane proteins contained uncleaved signal sequences which initiate translocation of the polypeptide chain. This review summarizes the central role that the signal peptides play as address codes for proteins, their decisive role as targeting factors for delivery to the membrane and their function to activate the translocation machinery for export and membrane protein insertion. After shedding light on the navigation of proteins, the importance of removal of signal peptide and their degradation are addressed. Furthermore, the emerging work on signal peptidases as novel targets for antibiotic development is described.

Bacterial Signal Peptides- Navigating the Journey of Proteins

The peptidisc membrane mimetic enables global reconstitution of the bacterial membrane proteome into water-soluble detergent-free particles, termed peptidisc libraries. We present here a method that combines peptidisc libraries and chromosomal-level gene tagging technology with affinity purification and mass spectrometry (AP/MS) to stabilize and identify fragile membrane protein complexes that exist at native expression levels. This method circumvents common artifacts caused by bait protein overproduction and protein complex dissociation due to lengthy exposure to detergents during protein isolation. Using the Escherichia coli Sec system as a case study, we identify an expanded version of the translocon, termed the HMD complex, consisting of nine different integral membrane subunits. This complex is stable in peptidiscs but dissociates in detergents. Guided by this native-level proteomic information, we design and validate a procedure that enables purification of the HMD complex with minimal protein dissociation. These results highlight the utility of peptidiscs and AP/MS to discover and stabilize fragile membrane protein assemblies. Data are available via ProteomeXchange with identifier PXD032315.

Development of a Method Combining Peptidiscs and Proteomics to Identify, Stabilize, and Purify a Detergent-Sensitive Membrane Protein Assembly.

The spatial and temporal coordination of protein transport is an essential cornerstone of the bacterial adaptation to different environmental conditions. By adjusting the protein composition of extra-cytosolic compartments, like the inner and outer membranes or the periplasmic space, protein transport mechanisms help shaping protein homeostasis in response to various metabolic cues. The universally conserved SecYEG translocon acts at the center of bacterial protein transport and mediates the translocation of newly synthesized proteins into and across the cytoplasmic membrane. The ability of the SecYEG translocon to transport an enormous variety of different substrates is in part determined by its ability to interact with multiple targeting factors, chaperones and accessory proteins. These interactions are crucial for the assisted passage of newly synthesized proteins from the cytosol into the different bacterial compartments. In this review, we summarize the current knowledge about SecYEG-mediated protein transport, primarily in the model organism Escherichia coli, and describe the dynamic interaction of the SecYEG translocon with its multiple partner proteins. We furthermore highlight how protein transport is regulated and explore recent developments in using the SecYEG translocon as an antimicrobial target.

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Robert Njenga

Papers

The Dynamic SecYEG Translocon.