In Vitro Reconstruction of Bacterial ß-Barrel Membrane Protein Assembly Using E. coli Microsomal (Mid-Density) Membrane.

The in vitro reconstruction assay enables us to evaluate in detail the insertion and proper protein folding (together termed assembly) of ß-barrel membrane proteins. Here, we introduce an in vitro reconstitution experiments using isolated membrane fractions from Escherichia coli (E. coli). Membrane fractions isolated from E. coli cells and disrupted by sonication, which we have termed E. coli microsomal (mid-density) membrane (EMM), are ideal for biochemical experiments, as they can be harvested by high-speed centrifugation and do not require ultra-centrifugation. EMM pretreated with detergent can assemble externally supplemented ß-barrel membrane proteins via intact ß-barrel assembly machinery (BAM) complex retained in EMM. This method not only allows assembly analysis with inexpensive equipment but it also can be applied to drug screening using assembly as an indicator with high reproducibility. In this chapter, we introduce our method of evaluating assembled ß-barrel membrane proteins by demonstrating four representative ß-barrel membrane proteins: E. coli major porins OmpA and OmpF; enterohemorrhagic E. coli (EHEC) autotransporter EspP, and Haemophilus influenzae (H. influenzae) adhesin Hia.

Dual recognition of multiple signals in bacterial outer membrane proteins enhances assembly and maintains membrane integrity.

Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a 'ß-signal' imprinted in the final ß-strand of the OMP engages the ß-barrel assembly machinery (BAM) complex to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule that signals equivalent to the ß-signal are repeated in other, internal ß-strands within bacterial OMPs, by peptidomimetic and mutational analysis. The internal signal is needed to promote the efficiency of the assembly reaction of these OMPs. BamD, an essential subunit of the BAM complex, recognizes the internal signal and the ß-signal, arranging several ß-strands and partial folding for rapid OMP assembly. The internal signal-BamD ordering system is not essential for bacterial viability but is necessary to retain the integrity of the outer membrane against antibiotics and other environmental insults.

Categorization of Escherichia coli outer membrane proteins by dependence on accessory proteins of the ß-barrel assembly machinery complex.

The outer membrane (OM) of gram-negative bacteria is populated by various outer membrane proteins (OMPs) that fold into a unique ß-barrel transmembrane domain. Most OMPs are assembled into the OM by the ß-barrel assembly machinery (BAM) complex. In Escherichia coli, the BAM complex is composed of two essential proteins (BamA and BamD) and three nonessential accessory proteins (BamB, BamC, and BamE). The currently proposed molecular mechanisms of the BAM complex involve only essential subunits, with the function of the accessory proteins remaining largely unknown. Here, we compared the accessory protein requirements for the assembly of seven different OMPs, 8- to 22-stranded, by our in vitro reconstitution assay using an E. coli mid-density membrane. BamE was responsible for the full efficiency of the assembly of all tested OMPs, as it enhanced the stability of essential subunit binding. BamB increased the assembly efficiency of more than 16-stranded OMPs, whereas BamC was not required for the assembly of any tested OMPs. Our categorization of the requirements of BAM complex accessory proteins in the assembly of substrate OMPs enables us to identify potential targets for the development of new antibiotics.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans.

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

The AAA ATPase Afg1 preserves mitochondrial fidelity and cellular health by maintaining mitochondrial matrix proteostasis.

Mitochondrial functions are critical for cellular physiology; therefore, several conserved mechanisms are in place to maintain the functional integrity of mitochondria. However, many of the molecular details and components involved in ensuring mitochondrial fidelity remain obscure. Here, we identify a novel role for the conserved mitochondrial AAA ATPase Afg1 in mediating mitochondrial protein homeostasis during aging and in response to various cellular challenges. Saccharomyces cerevisiae cells lacking functional Afg1 are hypersensitive to oxidative insults, unable to tolerate protein misfolding in the matrix compartment and exhibit progressive mitochondrial failure as they age. Loss of the Afg1 ortholog LACE-1 in Caenorhabditis elegans is associated with reduced lifespan, impeded oxidative stress tolerance, impaired mitochondrial proteostasis in the motor neuron circuitry and altered behavioral plasticity. Our results indicate that Afg1 is a novel protein quality control factor, which plays an important evolutionarily conserved role in mitochondrial surveillance, and cellular and organismal health.

The Assembly Factor Pet117 Couples Heme a Synthase Activity to Cytochrome Oxidase Assembly.

Heme a is an essential metalloporphyrin cofactor of the mitochondrial respiratory enzyme cytochrome c oxidase (CcO). Its synthesis from heme b requires several enzymes, including the evolutionarily conserved heme a synthase (Cox15). Oligomerization of Cox15 appears to be important for the process of heme a biosynthesis and transfer to maturing CcO. However, the details of this process remain elusive, and the roles of any additional CcO assembly factors that may be involved remain unclear. Here we report the systematic analysis of one such uncharacterized assembly factor, Pet117, and demonstrate in Saccharomyces cerevisiae that this evolutionarily conserved protein is necessary for Cox15 oligomerization and function. Pet117 is shown to reside in the mitochondrial matrix, where it is associated with the inner membrane. Pet117 functions at the later maturation stages of the core CcO subunit Cox1 that precede Cox1 hemylation. Pet117 also physically interacts with Cox15 and specifically mediates the stability of Cox15 oligomeric complexes. This Cox15-Pet117 interaction observed by co-immunoprecipitation persists in the absence of heme a synthase activity, is dependent upon Cox1 synthesis and early maturation steps, and is further dependent upon the presence of the matrix-exposed, unstructured linker region of Cox15 needed for Cox15 oligomerization, suggesting that this region mediates the interaction or that the interaction is lost when Cox15 is unable to oligomerize. Based on these findings, it was concluded that Pet117 mediates coupling of heme a synthesis to the CcO assembly process in eukaryotes.

Mitochondrial Quality Control Proteases in Neuronal Welfare.

The functional integrity of mitochondria is a critical determinant of neuronal health and compromised mitochondrial function is a commonly recognized factor that underlies a plethora of neurological and neurodegenerative diseases. Metabolic demands of neural cells require high bioenergetic outputs that are often associated with enhanced production of reactive oxygen species. Unopposed accumulation of these respiratory byproducts over time leads to oxidative damage and imbalanced protein homeostasis within mitochondrial subcompartments, which in turn may result in cellular demise. The post-mitotic nature of neurons and their vulnerability to these stress factors necessitate strict protein homeostatic control to prevent such scenarios. A series of evolutionarily conserved proteases is one of the central elements of mitochondrial quality control. These versatile proteolytic enzymes conduct a multitude of activities to preserve normal mitochondrial function during organelle biogenesis, metabolic remodeling and stress. In this review we discuss neuroprotective aspects of mitochondrial quality control proteases and neuropathological manifestations arising from defective proteolysis within the mitochondrion.

Theoretical and spectroscopic analysis of N,N'-diphenylurea and N,N'-dimethyl-N,N'-diphenylurea conformations.

Structural organization of macromolecules is highly dependent on the conformational propensity of the monomer units. Our goal is to systematically quantify differences in the conformational propensities of aromatic oligourea foldamer units. Specifically, we investigate the conformational propensities of N,N'-diphenylurea and N,N'-dimethyl-N,N'-diphenylurea in different media using a combination of theoretical methods, and infrared and nuclear magnetic resonance spectroscopies. Our results show variation in the conformational behavior upon adding methyl substituents on N,N'-diphenylurea, and varying the environments surrounding the compounds. Our energetic analyses and conformational distributions in the gas phase show predominance of the cis-trans and trans-trans conformations for N,N'-diphenylurea, while cis-cis conformation is favored for N,N'-dimethyl-N,N'-diphenylurea. In solution, our results support the trans-trans conformer as the predominant conformer for N,N'-diphenylurea, whereas the cis-cis and cis-trans forms are favored in N,N'-dimethyl-N,N'-diphenylurea. N,N'-Dimethyl-N,N'-diphenylurea also exhibits a more dynamic conformational behavior in solution, with constant fluctuations between cis-cis and cis-trans conformations. Our detailed quantitative analyses are an important aspect in fine-tuning desired conformations and dynamic properties of this class of oligomers by providing a molecular basis for the behavior at the monomeric level.