The β-Barrel Outer Membrane Protein Assembly Complex of Neisseria meningitidis

The evolutionarily conserved protein Omp85 is required for outer membrane protein (OMP) assembly in gram-negative bacteria and in mitochondria. Its Escherichia coli homolog, designated BamA, functions with four accessory lipoproteins, BamB, BamC, BamD, and BamE, together forming the β-barrel assembly machinery (Bam). Here, we addressed the composition of this machinery and the function of its components in Neisseria meningitidis, a model organism for outer membrane biogenesis studies. Analysis of genome sequences revealed homologs of BamC, BamD (previously described as ComL), and BamE and a second BamE homolog, Mlp. No homolog of BamB was found. As in E. coli, ComL/BamD appeared essential for viability and for OMP assembly, and it could not be replaced by its E. coli homolog. BamE was not essential but was found to contribute to the efficiency of OMP assembly and to the maintenance of OM integrity. A bamC mutant showed only marginal OMP assembly defects, but the impossibility of creating a bamC bamE double mutant further indicated the function of BamC in OMP assembly. An mlp mutant was unaffected in OMP assembly. The results of copurification assays demonstrated the association of BamC, ComL, and BamE with Omp85. Semi-native gel electrophoresis identified the RmpM protein as an additional component of the Omp85 complex, which was confirmed in copurification assays. RmpM was not required for OMP folding but stabilized OMP complexes. Thus, the Bam complex in N. meningitidis consists of Omp85/BamA plus RmpM, BamC, ComL/BamD, and BamE, of which ComL/BamD and BamE appear to be the most important accessory components for OMP assembly.Membrane-embedded β-barrel proteins are found in the outer membranes (OMs) of gram-negative bacteria, mitochondria, and chloroplasts. Only in recent years have cellular components required for the assembly and insertion of these OM proteins (OMPs) into the OM been identified. Omp85, which was first characterized in Neisseria meningitidis, is the key protein of the OMP assembly machinery (41). The function of Omp85 has been preserved during evolution, not only in gram-negative bacteria (8, 37, 44, 46) but also in mitochondria, where an Omp85 homolog, also known as Tob55 or Sam50, was shown to mediate the assembly of β-barrel proteins into the OM (15, 23, 27). Accordingly, bacterial OMPs are still recognized by the eukaryotic assembly machinery: when expressed in yeast, bacterial OMPs were found to be assembled into the mitochondrial OM in a Tob55-dependent manner (43). Omp85 in Escherichia coli, which was recently renamed BamA, for β-barrel assembly machinery (Bam) component A, is associated with at least four lipoproteins: BamB (formerly known as YfgL), BamC (NlpB), BamD (YfiO), and BamE (SmpA) (32, 46). In E. coli, BamB, BamC, and BamE are not essential, but the phenotypes of deletion mutants suggest that these proteins contribute to the efficiency of OMP assembly. Like BamA, BamD is an essential protein in E. coli (24, 26), involved in OMP assembly (24). These lipoproteins are evolutionarily less well conserved; the mitochondrial Tob55 protein is associated with two accessory proteins, but they do not show any sequence similarity with the lipoproteins of the E. coli Bam complex (14).Besides E. coli, N. meningitidis is one of the major bacterial model organisms for studies of OM assembly. As mentioned above, it was the first organism in which the function of Omp85 was identified (41), and also, the role of an integral OMP, designated LptD (formerly Imp or OstA), in the transport of lipopolysaccharide (LPS) to the cell surface was first established in N. meningitidis (3). With regard to OM biogenesis, N. meningitidis has several features that distinguish it from E. coli. For example, in contrast to E. coli (13), N. meningitidis mutants defective in LPS synthesis or transport are viable (3, 34), and OMPs are assembled perfectly well in such mutants (33). Furthermore, in OMP assembly mutants of E. coli, the periplasmic accumulation of unassembled OMPs is limited due to the induction of the σ^E extracytoplasmic stress response, which results in the degradation of unfolded OMPs (30) and the inhibition of their synthesis by small regulatory RNAs (20). In contrast, in N. meningitidis, most of the components involved in this response are absent (4), and unassembled OMPs continue to accumulate as periplasmic aggregates when OMP assembly is halted (41). However, the composition of the Bam complex and the role of accessory components in OMP assembly have not so far been studied in this organism. Therefore, to further understand the OMP assembly process in N. meningitidis, we have now analyzed the composition of the Bam complex and addressed the roles of the different components.     

Hemolytic Lectin CEL-III Heptamerizes via a Large Structural Transition from α-Helices to a β-Barrel during the Transmembrane Pore Formation Process

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer.     

pH-Dependent Interactions Guide the Folding and Gate the Transmembrane Pore of the β-Barrel Membrane Protein OmpG

The physical interactions that switch the functional state of membrane proteins are poorly understood. Previously, the pH-gating conformations of the β-barrel forming outer membrane protein G (OmpG) from Escherichia coli have been solved. When the pH changes from neutral to acidic the flexible extracellular loop L6 folds into and closes the OmpG pore. Here, we used single-molecule force spectroscopy to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At acidic pH, we detected a pH-dependent interaction at loop L6. This interaction changed the (un)folding of loop L6 and of β-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pH-dependent interactions change the folding of a peptide loop to gate the transmembrane pore. They further demonstrate how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore.     

The N-Terminal Transmembrane Domain of λ S Is Required for Holin but Not Antiholin Function

The λ S gene encodes a holin, S105, and an antiholin, S107, which differs by its Met-Lys N-terminal extension. The model for the lysis-defective character of S107 stipulates that the additional N-terminal basic residue keeps S107 from assuming the topology of S105, which is N-out, C-in, with three transmembrane domains (TMDs). Here we show that the N terminus of S105 retains its fMet residue but that the N terminus of S107 is fully deformylated. This supports the model that in S105, TMD1 inserts into the membrane very rapidly but that in S107, it is retained in the cytoplasm. Further, it reveals that, compared to S105, S107 has two extra positively charged moieties, Lys2 and the free N-terminal amino group, to hinder its penetration into an energized membrane. Moreover, an allele, S105_ΔTMD1, with TMD1 deleted, was found to be defective in lysis, insensitive to membrane depolarization, and dominant to the wild-type allele, indicating that the lysis-defective, antiholin character of S107 is due to the absence of TMD1 from the bilayer rather than to its ectopic localization at the inner face of the cytoplasmic membrane. Finally, the antiholin function of the deletion protein was compromised by the substitution of early-lysis missense mutations in either the deletion protein or parental S105 but restored when both S105_ΔTMD1 and holin carried the substitution.In general, holins control the length of the infection cycle of double-stranded DNA phages (37). During late gene expression, the holin protein accumulates harmlessly in the bilayer until suddenly and spontaneously triggering the formation of holes in the membrane at an allele-specific time (13, 15). Holin genes are extremely diverse, but most can be grouped into two main classes based on the number of predicted transmembrane domains (TMDs): class I, with three TMDs and a predicted N-out, C-in topology, and class II, with two TMDs and a predicted N-in, C-in topology (38). Holin genes and function are subject to several levels of regulation, among which a particularly striking feature is the common occurrence of two potential translational starts, or dual-start motifs (5, 37), separated by only a few codons. Dual-start motifs are found in many holins of both of the two major classes; in nearly every case, the two starts are separated by at least one basic residue. The first dual-start motif to be characterized was that of λ S, the prototype class I holin gene (Fig. 1A and B). Translation initiation events occur at codons 1 and 3, giving rise to two products, S107 and S105, each named because of the length of its amino acid sequence; in the wild-type (wt) allele, two RNA structures define the ratio of initiations at the two start codons, resulting in an S105/S107 ratio of ∼2:1.Open in a separate windowFIG. 1.Gene, topology, and sequence of λ S. (A, top) The λ lysis cassette, including genes S, R, Rz, and Rz1, is shown, along with its promoter p_R′, and Q, encoding the late gene activator. The 5′ end of the class I holin gene S has two start codons, Met1, the start for S107, and Met3, the start for S105, and two RNA structures that regulate initiations at these codons. The S105 and S107 alleles have Leu (CUG) codons in place of the Met3 and Met1 codons, respectively. (B) Primary structure of S proteins. Missense changes relevant to the text are shown. Starts for S107 and S105 are indicated by asterisks. The three TMDs are boxed (13), and the extent of the ΔTMD1 deletion is indicated. (C) Model for the membrane topology of S105, S107, and S105_ΔTMD1. Topology and boundary residues for TMD1, -2, and -3 are based on Graschopf and Blasi (11) and Gründling et al. (13), respectively.Although they differ only by the Met-Lys N-terminal extension of S107, the two proteins have opposing functions; S105 is the holin and S107 the antiholin. The antiholin function is reflected by four principal features: first, when the Met3 start is inactivated, the mutant allele, designated S107 (Fig. ​(Fig.1A),1A), is lysis defective (26); second, the S107 protein binds and inhibits S105 specifically (3, 16); third, when S107 is produced in stoichiometric excess over S105, lysis is blocked for several times the length of the normal infection cycle (3, 4, 7, 16); and fourth, S107 antiholin function, i.e., inhibition of S105 hole formation, can be instantly subverted by collapsing the proton motive force, most easily done by addition of energy poisons to the medium (3). The predicted N-out, C-in topology and the requirement for the energized membrane led to a model in which S107 is initially inserted in the membrane with only two TMDs, with TMD1 being blocked from insertion by the presence of the positively charged residue, Lys2, whereas S105 has three TMDs (Fig. ​(Fig.1C)1C) (39). From this perspective, S105-S107 complexes, which are approximately twice as numerous as the S105 homodimers, are defective in triggering hole formation. An appealing feature of this model is that when an S105-mediated hole formation event does occur in a cell, the resultant collapse of the membrane potential allows insertion of TMD1 of S107 into the membrane, instantly tripling the amount of active holin by making the previously inactive pool of S105-S107 complexes functional (38).Some genetic and physiological evidence for the topology of the λ S proteins has been obtained using gene fusions. First, a fusion of the S gene at codon 105 with lacZ generates a functional, membrane-inserted β-galactosidase chimera, indicating, as expected, the cytoplasmic disposition of the highly charged C terminus of the S protein (40). Second, Graschopf and Bläsi (12) demonstrated that S-mediated hole formation could be obtained with constructs where a secretory signal sequence was fused to the N termini of both S105 and S107. Lysis required the cleavage of the signal sequence by leader peptidase, and export of the signal-S107 form was slower than for the signal-S105 form. However, evidence for the topology of native forms of S has not been available. Moreover, no basis for the inhibitory character of S107 has been established. In the simplest view, the antiholin function could be due to the absence of TMD1 from the bilayer or the ectopic localization of TMD1 in the cytoplasm, or both. Here, we report studies directed at dissecting the precise role of topology in S107 function and correlating antiholin activity with its ability to heterodimerize with S105. The results are discussed in terms of a general model for the formation of the holin lesion and the role of dynamic membrane topology in its temporal regulation.     

The Small β-Barrel Domain: A Survey-Based Structural Analysis

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Folding of the β-Barrel Membrane Protein OmpA into Nanodiscs

Nanodiscs (NDs) are an excellent alternative to small unilamellar vesicles (SUVs) for studies of membrane protein structure, but it has not yet been shown that membrane proteins are able to spontaneously fold and insert into a solution of freely diffusing NDs. In this article, we present SDS-PAGE differential mobility studies combined with fluorescence, circular dichroism, and ultraviolet resonance Raman spectroscopy to confirm the spontaneous folding of outer membrane protein A (OmpA) into preformed NDs. Folded OmpA in NDs was incubated with Arg-C protease, resulting in the digestion of OmpA to membrane-protected fragments with an apparent molecular mass of ∼26 kDa (major component) and ∼24 kDa (minor component). The OmpA folding yields were greater than 88% in both NDs and SUVs. An OmpA adsorbed intermediate on NDs could be isolated at low temperature and induced to fold via an increase in temperature, analogous to the temperature-jump experiments on SUVs. The circular dichroism spectra of OmpA in NDs and SUVs were similar and indicated β-barrel secondary structure. Further evidence of OmpA folding into NDs was provided by ultraviolet resonance Raman spectroscopy, which revealed the intense 785 cm⁻¹ structural marker for folded OmpA in NDs. The primary difference between folding in NDs and SUVs was the kinetics; the rate of folding was two- to threefold slower in NDs compared to in SUVs, and this decreased rate can tentatively be attributed to the properties of NDs. These data indicate that NDs may be an excellent alternative to SUVs for folding experiments and offer benefits of optical clarity, sample homogeneity, control of ND:protein ratios, and greater stability.     

Pancreatic β-Cell Death due to Pdx-1 Deficiency Requires Multi-BH Domain Protein Bax but Not Bak

Diabetes develops in Pdx1-haploinsufficient mice due to an increase in β-cell death leading to reduced β-cell mass and decreased insulin secretion. Knockdown of Pdx1 gene expression in mouse MIN6 insulinoma cells induced apoptotic cell death with an increase in Bax activation and knockdown of Bax reduced apoptotic β-cell death. In Pdx1 haploinsufficient mice, Bax ablation in β-cells increased β-cell mass, decreased the number of TUNEL positive cells and improved glucose tolerance after glucose challenge. These changes were not observed with Bak ablation in Pdx1-haploinsufficient mice. These results suggest that Bax mediates β-cell apoptosis in Pdx1-deficient diabetes.     

The Protein-disulfide Isomerase DsbC Cooperates with SurA and DsbA in the Assembly of the Essential β-Barrel Protein LptD

The assembly of the β-barrel proteins present in the outer membrane (OM) of Gram-negative bacteria is poorly characterized. After translocation across the inner membrane, unfolded β-barrel proteins are escorted across the periplasm by chaperones that reside within this compartment. Two partially redundant chaperones, SurA and Skp, are considered to transport the bulk mass of β-barrel proteins. We found that the periplasmic disulfide isomerase DsbC cooperates with SurA and the thiol oxidase DsbA in the folding of the essential β-barrel protein LptD. LptD inserts lipopolysaccharides in the OM. It is also the only β-barrel protein with more than two cysteine residues. We found that surAdsbC mutants, but not skpdsbC mutants, exhibit a synthetic phenotype. They have a decreased OM integrity, which is due to the lack of the isomerase activity of DsbC. We also isolated DsbC in a mixed disulfide complex with LptD. As such, LptD is identified as the first substrate of DsbC that is localized in the OM. Thus, electrons flowing from the cytoplasmic thioredoxin system maintain the integrity of the OM by assisting the folding of one of the most important β-barrel proteins.     

Brain Acetylcholinesterase Promotes Amyloid-β-Peptide Aggregation but Does Not Hydrolyze Amyloid Precursor Protein Peptides

It has been suggested that acetylcholinesterase (AChE) has both a putative proteolytic activity against the amyloid precursor protein (APP), and a capacity to accelerate the assembly of amyloid--peptide (A) into Alzheimer's fibrils. Here, we have studied the ability of bovine brain AChE to share both activities. Results indicate that AChE purified through acridinium was able to process the APP peptides, however after further purification by an edrophonium column, the protease activity was lost. Under both conditions the capacity of the enzyme to promote amyloid formation was maintained. Kinetic studies of the A aggregation process using edrophonium-AChE, indicated that the lag phase of the aggregation process was smaller than the one observed with the esterase purified by acridinium alone. Considering that the total amount of amyloid formed, measured by thioflavine-T fluorescence, was similar for both AChE preparations, our results suggest that the edrophonium-AChE possesses an higher intrinsic capacity to stimulate the aggregation of A_1–40 peptide.     

The Vitamin D Analogue ED71 but Not 1,25(OH)2D3 Targets HIF1α Protein in Osteoclasts

Although both an active form of the vitamin D metabolite, 1,25(OH)₂D₃, and the vitamin D analogue, ED71 have been used to treat osteoporosis, anti-bone resorbing activity is reportedly seen only in ED71- but not in 1,25(OH)₂D₃ -treated patients. In addition, how ED71 inhibits osteoclast activity in patients has not been fully characterized. Recently, HIF1α expression in osteoclasts was demonstrated to be required for development of post-menopausal osteoporosis. Here we show that ED71 but not 1,25(OH)₂D₃, suppress HIF1α protein expression in osteoclasts in vitro. We found that 1,25(OH)₂D₃ or ED71 function in osteoclasts requires the vitamin D receptor (VDR). ED71 was significantly less effective in inhibiting M-CSF and RANKL-stimulated osteoclastogenesis than was 1,25(OH)₂D₃in vitro. Downregulation of c-Fos protein and induction of Ifnβ mRNA in osteoclasts, both of which reportedly block osteoclastogenesis induced by 1,25(OH)₂D₃in vitro, were both significantly higher following treatment with 1,25(OH)₂D₃ than with ED71. Thus, suppression of HIF1α protein activity in osteoclasts in vitro, which is more efficiently achieved by ED71 rather than by 1,25(OH)₂D₃, could be a reliable read-out in either developing or screening reagents targeting osteoporosis.     

Protein Kinase A Activation Phosphorylates the Rat ClC-2 Cl− Channel but Does Not Change Activity

Phosphorylation-dependent events have been shown to modulate the activity of several members of the mammalian CLC Cl⁻ channel gene family, including the inward rectifier ClC-2. In the present study we investigated the regulation of rat ClC-2 expressed in the TSA-201 cell line (a transformed HEK293 cell line that stably expresses the SV40 T-antigen) by protein kinases. Protein kinase A activation phosphorylated ClC-2 in vivo, whereas stimulation of protein kinase C with phorbol 12-myristate 13-acetate did not. In vitro labeling studies confirmed that protein kinase A could directly phosphorylate ClC-2, and that protein kinase C and Ca²⁺/calmodulin-dependent protein kinase II did not. Nevertheless, protein kinase A-dependent phosphorylation of CLC-2 failed to regulate either the magnitude or the kinetics of the hyperpolarization-activated Cl⁻ currents. Considered together, we demonstrate that protein kinase A activation results in the phosphorylation of rat ClC-2 in vivo, but this event is independent of Cl⁻ channel activity. Received: 20 November 2000/Revised: 28 March 2001     

Phosphorylated Ribosomal Protein S6 Is Required for Akt-Driven Hyperplasia and Malignant Transformation,but Not for Hypertrophy,Aneuploidy and Hyperfunction of Pancreatic β-Cells

Constitutive expression of active Akt (Akt^tg) drives hyperplasia and hypertrophy of pancreatic β-cells, concomitantly with increased insulin secretion and improved glucose tolerance, and at a later stage the development of insulinoma. To determine which functions of Akt are mediated by ribosomal protein S6 (rpS6), an Akt effector, we generated mice that express constitutive Akt in β-cells in the background of unphosphorylatable ribosomal protein S6 (rpS6^P-/-). rpS6 phosphorylation deficiency failed to block Akt^tg-induced hypertrophy and aneuploidy in β-cells, as well as the improved glucose homeostasis, indicating that Akt carries out these functions independently of rpS6 phosphorylation. In contrast, rpS6 phosphorylation deficiency efficiently restrained the reduction in nuclear localization of the cell cycle inhibitor p27, as well as the development of Akt^tg-driven hyperplasia and tumor formation in β-cells. In vitro experiments with Akt^tg and rpS6^P-/-;Akt^tg fibroblasts demonstrated that rpS6 phosphorylation deficiency leads to reduced translation fidelity, which might underlie its anti-tumorigenic effect in the pancreas. However, the role of translation infidelity in tumor suppression cannot simply be inferred from this heterologous experimental model, as rpS6 phosphorylation deficiency unexpectedly elevated the resistance of Akt^tg fibroblasts to proteotoxic, genotoxic as well as autophagic stresses. In contrast, rpS6^P-/- fibroblasts exhibited a higher sensitivity to these stresses upon constitutive expression of oncogenic Kras. The latter result provides a possible mechanistic explanation for the ability of rpS6 phosphorylation deficiency to enhance DNA damage and protect mice from Kras-induced neoplastic transformation in the exocrine pancreas. We propose that Akt1 and Kras exert their oncogenic properties through distinct mechanisms, even though both show addiction to rpS6 phosphorylation.     

‘The Earth Cannot Let Go of Us’: Analysing Ontological Conflicts

This paper examines certain key concepts of the ‘ontological turn’ in anthropology, with a view to a clearer understanding of its proposed methodology. It situates the ontological approach within the historical and intellectual conflicts through which it arose, outlining its motivations and the challenges it poses to traditional fieldwork methods and theory. The concepts of ‘radical alterity’ and ‘incommensurability’ are examined as intellectual as well as political concepts, highlighting their historical contingency on the politics of colonisation. Following from this, the notion of ‘ontological self-determination’ is analysed with respect to my fieldwork with the Dongria Kondh in the Niyamgiri Hills in Odisha, India; I show how an application of the ontological methodology allows a better understanding of certain conflicts within current discourses and practices of nationhood and development.     

The Roles of Protein Kinase C βI and βII in Vascular Smooth Muscle Cell Proliferation

The role of protein kinase C (PKC) on proliferation of A10 vascular smooth muscle cells (VSMC) was studied by overexpressing specific PKC-βI and -βII isozymes. PKC-βI and -βII are derived from alternative splicing of the exon encoding the carboxy-terminal (C-terminal) 50 or 52 amino acids, respectively. The differential functions of the two isozymes with regard to cell proliferation, DNA synthesis, and the cell cycle were investigated in A10 cells, a clonal cell line of VSMC from rat aorta, and in A10 cells overexpressing PKC-βI and PKC-βII (βI-A10 and βII-A10). PKC levels were increased three- to fourfold in heterogeneous cultures of stably transfected cells. Although doubling time of A10 cells was 36 h, the cell doubling time in βI-A10 cells decreased by 12 h, and, in contrast, the doubling time of βII-A10 cells increased by 12 h compared to A10 cells. The increase of [³H]thymidine (TdR) incorporation was accelerated and increased in βI-A10 cells, but slowed and diminished in βII-A10 cells compared to A10 and control cells transfected with empty vector. Cell cycle analysis of βI-A10 cells showed an acceleration of S phase entry while βII-A10 cells slowed S phase entry. These results suggest that PKC-βI and PKC-βII regulate cell proliferation bidirectionally and that PKC-βI and PKC-βII may have distinct and opposing functions as cell cycle check point mediators during late G₁phase and may regulate S phase entry in A10 VSMC.     

Analysis of Mutations in Neurospora crassa ERMES Components Reveals Specific Functions Related to β-Barrel Protein Assembly and Maintenance of Mitochondrial Morphology

The endoplasmic reticulum mitochondria encounter structure (ERMES) tethers the ER to mitochondria and contains four structural components: Mmm1, Mdm12, Mdm10, and Mmm2 (Mdm34). The Gem1 protein may play a role in regulating ERMES function. Saccharomyces cerevisiae and Neurospora crassa strains lacking any of Mmm1, Mdm12, or Mdm10 are known to show a variety of phenotypic defects including altered mitochondrial morphology and defects in the assembly of β-barrel proteins into the mitochondrial outer membrane. Here we examine ERMES complex components in N. crassa and show that Mmm1 is an ER membrane protein containing a Cys residue near its N-terminus that is conserved in the class Sordariomycetes. The residue occurs in the ER-lumen domain of the protein and is involved in the formation of disulphide bonds that give rise to Mmm1 dimers. Dimer formation is required for efficient assembly of Tom40 into the TOM complex. However, no effects are seen on porin assembly or mitochondrial morphology. This demonstrates a specificity of function and suggests a direct role for Mmm1 in Tom40 assembly. Mutation of a highly conserved region in the cytosolic domain of Mmm1 results in moderate defects in Tom40 and porin assembly, as well as a slight morphological phenotype. Previous reports have not examined the role of Mmm2 with respect to mitochondrial protein import and assembly. Here we show that absence of Mmm2 affects assembly of β-barrel proteins and that lack of any ERMES structural component results in defects in Tom22 assembly. Loss of N. crassa Gem1 has no effect on the assembly of these proteins but does affect mitochondrial morphology.