A virus capsid‐like nanocompartment that stores iron and protects bacteria from oxidative stress

Living cells compartmentalize materials and enzymatic reactions to increase metabolic efficiency. While eukaryotes use membrane‐bound organelles, bacteria and archaea rely primarily on protein‐bound nanocompartments. Encapsulins constitute a class of nanocompartments widespread in bacteria and archaea whose functions have hitherto been unclear. Here, we characterize the encapsulin nanocompartment from Myxococcus xanthus, which consists of a shell protein (EncA, 32.5 kDa) and three internal proteins (EncB, 17 kDa; EncC, 13 kDa; EncD, 11 kDa). Using cryo‐electron microscopy, we determined that EncA self‐assembles into an icosahedral shell 32 nm in diameter (26 nm internal diameter), built from 180 subunits with the fold first observed in bacteriophage HK97 capsid. The internal proteins, of which EncB and EncC have ferritin‐like domains, attach to its inner surface. Native nanocompartments have dense iron‐rich cores. Functionally, they resemble ferritins, cage‐like iron storage proteins, but with a massively greater capacity (~30,000 iron atoms versus ~3,000 in ferritin). Physiological data reveal that few nanocompartments are assembled during vegetative growth, but they increase fivefold upon starvation, protecting cells from oxidative stress through iron sequestration.     

The protein shells of bacterial microcompartment organelles

Details are emerging on the structure and function of a remarkable class of capsid-like protein assemblies that serve as simple metabolic organelles in many bacteria. These bacterial microcompartments consist of a few thousand shell proteins, which encapsulate two or more sequentially acting enzymes in order to enhance or sequester certain metabolic pathways, particularly those involving toxic or volatile intermediates. Genomic data indicate that bacterial microcompartment shell proteins are present in a wide range of bacterial species, where they encapsulate varied reactions. Crystal structures of numerous shell proteins from distinct types of microcompartments have provided keys for understanding how the shells are assembled and how they conduct molecular transport into and out of microcompartments. The structural data emphasize a high level of mechanistic sophistication in the protein shell, and point the way for further studies on this fascinating but poorly appreciated class of subcellular structures.     

De novo design of signal sequences to localize cargo to the 1,2‐propanediol utilization microcompartment

Organizing heterologous biosyntheses inside bacterial cells can alleviate common problems owing to toxicity, poor kinetic performance, and cofactor imbalances. A subcellular organelle known as a bacterial microcompartment, such as the 1,2‐propanediol utilization microcompartment of Salmonella, is a promising chassis for this strategy. Here we demonstrate de novo design of the N‐terminal signal sequences used to direct cargo to these microcompartment organelles. We expand the native repertoire of signal sequences using rational and library‐based approaches and show that a canonical leucine‐zipper motif can function as a signal sequence for microcompartment localization. Our strategy can be applied to generate new signal sequences localizing arbitrary cargo proteins to the 1,2‐propanediol utilization microcompartments.     

Exploiting genomic patterns to discover new supramolecular protein assemblies

Bacterial microcompartments are supramolecular protein assemblies that function as bacterial organelles by compartmentalizing particular enzymes and metabolic intermediates. The outer shells of these microcompartments are assembled from multiple paralogous structural proteins. Because the paralogs are required to assemble together, their genes are often transcribed together from the same operon, giving rise to a distinctive genomic pattern: multiple, typically small, paralogous proteins encoded in close proximity on the bacterial chromosome. To investigate the generality of this pattern in supramolecular assemblies, we employed a comparative genomics approach to search for protein families that show the same kind of genomic pattern as that exhibited by bacterial microcompartments. The results indicate that a variety of large supramolecular assemblies fit the pattern, including bacterial gas vesicles, bacterial pili, and small heat‐shock protein complexes. The search also retrieved several widely distributed protein families of presently unknown function. The proteins from one of these families were characterized experimentally and found to show a behavior indicative of supramolecular assembly. We conclude that cotranscribed paralogs are a common feature of diverse supramolecular assemblies, and a useful genomic signature for discovering new kinds of large protein assemblies from genomic data.     

Protein-based organelles in bacteria: carboxysomes and related microcompartments

Many bacteria contain intracellular microcompartments with outer shells that are composed of thousands of protein subunits and interiors that are filled with functionally related enzymes. These microcompartments serve as organelles by sequestering specific metabolic pathways in bacterial cells. The carboxysome, a prototypical bacterial microcompartment that is found in cyanobacteria and some chemoautotrophs, encapsulates ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase, and thereby enhances carbon fixation by elevating the levels of CO2 in the vicinity of RuBisCO. Evolutionarily related, but functionally distinct, microcompartments are present in diverse bacteria. Although bacterial microcompartments were first observed more than 40 years ago, a detailed understanding of how they function is only now beginning to emerge.     

Advances in encapsulin nanocompartment biology and engineering

Compartmentalization is an essential feature of all cells. It allows cells to segregate and coordinate physiological functions in a controlled and ordered manner. Different mechanisms of compartmentalization exist, with the most relevant to prokaryotes being encapsulation via self‐assembling protein‐based compartments. One widespread example of such is that of encapsulins—cage‐like protein nanocompartments able to compartmentalize specific reactions, pathways, and processes in bacteria and archaea. While still relatively nascent bioengineering tools, encapsulins exhibit many promising characteristics, including a number of defined compartment sizes ranging from 24 to 42 nm, straightforward expression, the ability to self‐assemble via the Hong Kong 97‐like fold, marked physical robustness, and internal and external handles primed for rational genetic and molecular manipulation. Moreover, encapsulins allow for facile and specific encapsulation of native or heterologous cargo proteins via naturally or rationally fused targeting peptide sequences. Taken together, the attributes of encapsulins promise substantial customizability and broad usability. This review discusses recent advances in employing engineered encapsulins across various fields, from their use as bionanoreactors to targeted delivery systems and beyond. A special focus will be provided on the rational engineering of encapsulin systems and their potential promise as biomolecular research tools.     

Using comparative genomics to uncover new kinds of protein‐based metabolic organelles in bacteria

Bacterial microcompartment (MCP) organelles are cytosolic, polyhedral structures consisting of a thin protein shell and a series of encapsulated, sequentially acting enzymes. To date, different microcompartments carrying out three distinct types of metabolic processes have been characterized experimentally in various bacteria. In the present work, we use comparative genomics to explore the existence of yet uncharacterized microcompartments encapsulating a broader set of metabolic pathways. A clustering approach was used to group together enzymes that show a strong tendency to be encoded in chromosomal proximity to each other while also being near genes for microcompartment shell proteins. The results uncover new types of putative microcompartments, including one that appears to encapsulate B₁₂‐independent, glycyl radical‐based degradation of 1,2‐propanediol, and another potentially involved in amino alcohol metabolism in mycobacteria. Preliminary experiments show that an unusual shell protein encoded within the glycyl radical‐based microcompartment binds an iron‐sulfur cluster, hinting at complex mechanisms in this uncharacterized system. In addition, an examination of the computed microcompartment clusters suggests the existence of specific functional variations within certain types of MCPs, including the alpha carboxysome and the glycyl radical‐based microcompartment. The findings lead to a deeper understanding of bacterial microcompartments and the pathways they sequester.     

Bacterial microcompartments: widespread prokaryotic organelles for isolation and optimization of metabolic pathways

Prokaryotes use subcellular compartments for a variety of purposes. An intriguing example is a family of complex subcellular organelles known as bacterial microcompartments (MCPs). MCPs are widely distributed among bacteria and impact processes ranging from global carbon fixation to enteric pathogenesis. Overall, MCPs consist of metabolic enzymes encased within a protein shell, and their function is to optimize biochemical pathways by confining toxic or volatile metabolic intermediates. MCPs are fundamentally different from other organelles in having a complex protein shell rather than a lipid‐based membrane as an outer barrier. This unusual feature raises basic questions about organelle assembly, protein targeting and metabolite transport. In this review, we discuss the three best‐studied MCPs highlighting atomic‐level models for shell assembly, targeting sequences that direct enzyme encapsulation, multivalent proteins that organize the lumen enzymes, the principles of metabolite movement across the shell, internal cofactor recycling, a potential system of allosteric regulation of metabolite transport and the mechanism and rationale behind the functional diversification of the proteins that form the shell. We also touch on some potential biotechnology applications of an unusual compartment designed by nature to optimize metabolic processes within a cellular context.     

Elucidating essential role of conserved carboxysomal protein CcmN reveals common feature of bacterial microcompartment assembly

Bacterial microcompartments are organelles composed of a protein shell that surrounds functionally related proteins. Bioinformatic analysis of sequenced genomes indicates that homologs to shell protein genes are widespread among bacteria and suggests that the shell proteins are capable of encapsulating diverse enzymes. The carboxysome is a bacterial microcompartment that enhances CO(2) fixation in cyanobacteria and some chemoautotrophs by sequestering ribulose-1,5-bisphosphate carboxylase/oxygenase and carbonic anhydrase in the microcompartment shell. Here, we report the in vitro and in vivo characterization of CcmN, a protein of previously unknown function that is absolutely conserved in β-carboxysomal gene clusters. We show that CcmN localizes to the carboxysome and is essential for carboxysome biogenesis. CcmN has two functionally distinct regions separated by a poorly conserved linker. The N-terminal portion of the protein is important for interaction with CcmM and, by extension, ribulose-1,5-bisphosphate carboxylase/oxygenase and the carbonic anhydrase CcaA, whereas the C-terminal peptide is essential for interaction with the carboxysome shell. Deletion of the peptide abolishes carboxysome formation, indicating that its interaction with the shell is an essential step in microcompartment formation. Peptides with similar length and sequence properties to those in CcmN can be bioinformatically detected in a large number of diverse proteins proposed to be encapsulated in functionally distinct microcompartments, suggesting that this peptide and its interaction with its cognate shell proteins are common features of microcompartment assembly.     

Probe into a multi-protein prokaryotic organelle using thermal scanning assay reveals distinct properties of the core and the shell

BackgroundBacterial microcompartments represent the only reported category of prokaryotic organelles that are capable of functioning as independent bioreactors. In this organelle, a biochemical pathway with all the enzyme machinery is encapsulated within an all protein shell. The shell proteins and the enzymes have distinct structural features. It is hypothesized that flat shell proteins align sideways to form extended sheets and, the globular enzymes fill up the central core of the organelle.MethodsUsing differential scanning fluorimetry, we explored the structure and functional alteration of Pdu BMC, involving tertiary or quaternary structures.ResultsOur findings exhibit that these intact BMCs as a whole behave similar to a globular protein with a rich hydrophobic core, which is exposed upon thermal insult. The encapsulated enzymes itself have a strong hydrophobic core, which is in line with the hydrophobic-collapse model of protein folding. The shell proteins, on the other hand, do not have a strong hydrophobic core and show a significant portion of exposed hydrophobic patches.ConclusionWe show for the first time the thermal unfolding profile of the BMC domain proteins and the unique exposure of hydrophobic patches in them might be required for anchoring the enzymes leading to better packaging of the micro-compartments.General significanceThese observations indicate that the genesis of these unique bacterial organelles is driven by the hydrophobic interactions between the shell and the enzymes. Insights from this work will aid in the genetic and biochemical engineering of thermostable efficient enzymatic biomaterials.     

Two new high-resolution crystal structures of carboxysome pentamer proteins reveal high structural conservation of CcmL orthologs among distantly related cyanobacterial species

Cyanobacteria have evolved a unique carbon fixation organelle known as the carboxysome that compartmentalizes the enzymes RuBisCO and carbonic anhydrase. This effectively increases the local CO₂ concentration at the active site of RuBisCO and decreases its relatively unproductive side reaction with oxygen. Carboxysomes consist of a protein shell composed of hexameric and pentameric proteins arranged in icosahedral symmetry. Facets composed of hexameric proteins are connected at the vertices by pentameric proteins. Structurally homologous pentamers and hexamers are also found in heterotrophic bacteria where they form architecturally related microcompartments such as the Eut and Pdu organelles for the metabolism of ethanolamine and propanediol, respectively. Here we describe two new high-resolution structures of the pentameric shell protein CcmL from the cyanobacteria Thermosynechococcus elongatus and Gloeobacter violaceus and provide detailed analysis of their characteristics and comparison with related shell proteins.     

Longin-like folds identified in CHiPS and DUF254 proteins: vesicle trafficking complexes conserved in eukaryotic evolution

Eukaryotic protein trafficking pathways require specific transfer of cargo vesicles to different target organelles. A number of vesicle trafficking and membrane fusion components participate in this process, including various tethering factor complexes that interact with small GTPases prior to SNARE-mediated vesicle fusion. In Saccharomyces cerevisiae a protein complex of Mon1 and Ccz1 functions with the small GTPase Ypt7 to mediate vesicle trafficking to the vacuole. Mon1 belongs to DUF254 found in a diverse range of eukaryotic genomes, while Ccz1 includes a CHiPS domain that is also present in a known human protein trafficking disorder gene (HPS-4). The present work identifies the CHiPS domain and a sequence region from another trafficking disorder gene (HPS-1) as homologs of an N-terminal domain from DUF254. This link establishes the evolutionary conservation of a protein complex (HPS-1/HPS-4) that functions similarly to Mon1/Ccz1 in vesicle trafficking to lysosome-related organelles of diverse eukaryotic species. Furthermore, the newly identified DUF254 domain is a distant homolog of the mu-adaptin longin domain found in clathrin adapter protein (AP) complexes of known structure that function to localize cargo protein to specific organelles. In support of this fold assignment, known longin domains such as the AP complex sigma-adaptin, the synaptobrevin N-terminal domains sec22 and Ykt6, and the srx domain of the signal recognition particle receptor also regulate vesicle trafficking pathways by mediating SNARE fusion, recognizing specialized compartments, and interacting with small GTPases that resemble Ypt7.     

SPdb – a signal peptide database

Background  

The signal peptide plays an important role in protein targeting and protein translocation in both prokaryotic and eukaryotic cells. This transient, short peptide sequence functions like a postal address on an envelope by targeting proteins for secretion or for transfer to specific organelles for further processing. Understanding how signal peptides function is crucial in predicting where proteins are translocated. To support this understanding, we present SPdb signal peptide database , a repository of experimentally determined and computationally predicted signal peptides.     

Bacterial microcompartments: their properties and paradoxes

Many bacteria conditionally express proteinaceous organelles referred to here as microcompartments (Fig. 1). These microcompartments are thought to be involved in a least seven different metabolic processes and the number is growing. Microcompartments are very large and structurally sophisticated. They are usually about 100-150 nm in cross section and consist of 10,000-20,000 polypeptides of 10-20 types. Their unifying feature is a solid shell constructed from proteins having bacterial microcompartment (BMC) domains. In the examples that have been studied, the microcompartment shell encases sequentially acting metabolic enzymes that catalyze a reaction sequence having a toxic or volatile intermediate product. It is thought that the shell of the microcompartment confines such intermediates, thereby enhancing metabolic efficiency and/or protecting cytoplasmic components. Mechanistically, however, this creates a paradox. How do microcompartments allow enzyme substrates, products and cofactors to pass while confining metabolic intermediates in the absence of a selectively permeable membrane? We suggest that the answer to this paradox may have broad implications with respect to our understanding of the fundamental properties of biological protein sheets including microcompartment shells, S-layers and viral capsids.     

Self-assembling Shell Proteins PduA and PduJ have Essential and Redundant Roles in Bacterial Microcompartment Assembly

Protein self-assembly is a common and essential biological phenomenon, and bacterial microcompartments present a promising model system to study this process. Bacterial microcompartments are large, protein-based organelles which natively carry out processes important for carbon fixation in cyanobacteria and the survival of enteric bacteria. These structures are increasingly popular with biological engineers due to their potential utility as nanobioreactors or drug delivery vehicles. However, the limited understanding of the assembly mechanism of these bacterial microcompartments hinders efforts to repurpose them for non-native functions. Here, we comprehensively investigate proteins involved in the assembly of the 1,2-propanediol utilization bacterial microcompartment from Salmonella enterica serovar Typhimurium LT2, one of the most widely studied microcompartment systems. We first demonstrate that two shell proteins, PduA and PduJ, have a high propensity for self-assembly upon overexpression, and we provide a novel method for self-assembly quantification. Using genomic knock-outs and knock-ins, we systematically show that these two proteins play an essential and redundant role in bacterial microcompartment assembly that cannot be compensated by other shell proteins. At least one of the two proteins PduA and PduJ must be present for the bacterial microcompartment shell to assemble. We also demonstrate that assembly-deficient variants of these proteins are unable to rescue microcompartment formation, highlighting the importance of this assembly property. Our work provides insight into the assembly mechanism of these bacterial organelles and will aid downstream engineering efforts.     

New directions for the clathrin adaptor AP-1 in cell biology and human disease

The clathrin adaptor protein complex-1 (AP-1) is a central player in cell physiology and human health. It is best known for its role in linking clathrin to its cargo at the trans-Golgi network and endosomes. It participates in traffic important for the correct function of a large number of organelles, including the trans-Golgi network, endosomes, lysosomes, lysosome-related organelles, and plasma membrane. Although it was one of the first clathrin adaptors identified, new discoveries about cargo and pathways that depend on AP-1 continue to emerge. This review summarizes new research into AP-1 that further illuminates its roles in the traffic of plasma membrane proteins, in maintaining TGN content, and in human disease.     

Lipid-transfer proteins in membrane trafficking at the Golgi complex

The Golgi complex (GC) represents the central junction for membrane trafficking. Protein and lipid cargoes continuously move through the GC in both anterograde and retrograde directions, departing to and arriving from diverse destinations within the cell. Nevertheless, the GC is able to maintain its identity and strict compartmentalisation, having a different composition in terms of protein and lipid content compared to other organelles. The discovery of coat protein complexes and the elucidation of their role in sorting cargo proteins into specific transport carriers have provided a partial answer to this phenomenon. However, it is more difficult to understand how relatively small and diffusible molecules like lipids can be concentrated in or excluded from specific subcellular compartments. The discovery of lipid-transfer proteins operating in the secretory pathway and specifically at the GC has shed light on one possible way in which this lipid compartmentalisation can be accomplished. The correct lipid distribution along the secretory pathway is of crucial importance for cargo protein sorting and secretion. This review focuses on what is now known about the putative and effective lipid-transfer proteins at the GC, and on how they affect the function and structure of the GC itself.     

Two MAPK-signaling pathways are required for mitophagy in Saccharomyces cerevisiae

Macroautophagy (hereafter referred to simply as autophagy) is a catabolic pathway that mediates the degradation of long-lived proteins and organelles in eukaryotic cells. The regulation of mitochondrial degradation through autophagy plays an essential role in the maintenance and quality control of this organelle. Compared with our understanding of the essential function of mitochondria in many aspects of cellular metabolism such as energy production and of the role of dysfunctional mitochondria in cell death, little is known regarding their degradation and especially how upstream signaling pathways control this process. Here, we report that two mitogen-activated protein kinases (MAPKs), Slt2 and Hog1, are required for mitophagy in Saccharomyces cerevisiae. Slt2 is required for the degradation of both mitochondria and peroxisomes (via pexophagy), whereas Hog1 functions specifically in mitophagy. Slt2 also affects the recruitment of mitochondria to the phagophore assembly site (PAS), a critical step in the packaging of cargo for selective degradation.     

Polyhedral organelles compartmenting bacterial metabolic processes

Bacterial polyhedral organelles are extremely large macromolecular complexes consisting of metabolic enzymes encased within a multiprotein shell that is somewhat reminiscent of a viral capsid. Recent investigations suggest that polyhedral organelles are widely used by bacteria for optimizing metabolic processes. The distribution and diversity of these unique structures has been underestimated because many are not formed during growth on standard laboratory media and because electron microscopy is required for their observation. However, recent physiological studies and genomic analyses tentatively indicate seven functionally distinct organelles distributed among over 40 genera of bacteria. Functional studies conducted thus far are consistent with the idea that polyhedral organelles act as microcompartments that enhance metabolic processes by selectively concentrating specific metabolites. Relatively little is known about how this is achieved at the molecular level. Possible mechanisms include regulation of enzyme activity or efficiency, substrate channeling, a selectively permeable protein shell, and/or differential solubility of metabolites within the organelle. Given their complexity and distinctive structure, it would not be surprising if aspects of their biochemical mechanism are unique. Therefore, the unusual structure of polyhedral organelles raises intriguing questions about their assembly, turnover, and molecular evolution, very little of which is understood.     

Analysis of the Structure of the PsbO Protein and its Implications

The PsbO protein is a ubiquitous extrinsic subunit of Photosystem II (PS II), the water splitting enzyme of photosynthesis. A recently determined 3D X-ray structure of a cyanobacterial protein bound to PS II has given an opportunity to conduct complete analyses of its sequence and structural characteristics using bioinformatic methods. Multiple sequence alignments for the PsbO family are constructed and correlated with the cyanobacterial structure. We identify the most conserved regions of PsbO and the mapping of their positions within the structure indicates their functional roles especially in relation to interactions of this protein with the lumenal surface of PS II. Homologous models for eukaryotic PsbO were built in order to compare with the prokaryotic protein. We also explore structural homology between PsbO and other proteins for which 3D structures are known and determine its structural classification. These analyses contribute to the understanding of the function and evolutionary origin of the PS II manganese stabilising protein.