Outer membrane protein biogenesis in Gram-negative bacteria

Gram-negative bacteria contain a double membrane which serves for both protection and for providing nutrients for viability. The outermost of these membranes is called the outer membrane (OM), and it contains a host of fully integrated membrane proteins which serve essential functions for the cell, including nutrient uptake, cell adhesion, cell signalling and waste export. For pathogenic strains, many of these outer membrane proteins (OMPs) also serve as virulence factors for nutrient scavenging and evasion of host defence mechanisms. OMPs are unique membrane proteins in that they have a β-barrel fold and can range in size from 8 to 26 strands, yet can still serve many different functions for the cell. Despite their essential roles in cell survival and virulence, the exact mechanism for the biogenesis of these OMPs into the OM has remained largely unknown. However, the past decade has witnessed significant progress towards unravelling the pathways and mechanisms necessary for moulding a nascent polypeptide into a functional OMP within the OM. Here, we will review some of these recent discoveries that have advanced our understanding of the biogenesis of OMPs in Gram-negative bacteria, starting with synthesis in the cytoplasm to folding and insertion into the OM.     

Peroxisome assembly: matrix and membrane protein biogenesis

Mitochondria are dynamic organelles whose functional integrity requires a coordinated supply of proteins and phospholipids. Defined functions of specific phospholipids, like the mitochondrial signature lipid cardiolipin, are emerging in diverse processes, ranging from protein biogenesis and energy production to membrane fusion and apoptosis. The accumulation of phospholipids within mitochondria depends on interorganellar lipid transport between the endoplasmic reticulum (ER) and mitochondria as well as intramitochondrial lipid trafficking. The discovery of proteins that regulate mitochondrial membrane lipid composition and of a multiprotein complex tethering ER to mitochondrial membranes has unveiled novel mechanisms of mitochondrial membrane biogenesis.     

Versatility of inner membrane protein biogenesis in Escherichia coli

To further our understanding of inner membrane protein (IMP) biogenesis in Escherichia coli, we have accomplished the widest in vivo IMP assembly screen so far. The biogenesis of a set of model IMPs covering most IMP structures possible has been studied in a variety of signal recognition particle (SRP), Sec and YidC mutant strains. We show that the assembly of the complete set of model IMPs is assisted (i.e. requires the aid of proteinaceous factors), and that the requirements for assembly of the model IMPs into the inner membrane differ significantly from each other. This indicates that IMP assembly is much more versatile than previously thought.     

Flexibility in targeting and insertion during bacterial membrane protein biogenesis

The biogenesis of Escherichia coli inner membrane proteins (IMPs) is assisted by targeting and insertion factors such as the signal recognition particle (SRP), the Sec-translocon and YidC with translocation of (large) periplasmic domains energized by SecA and the proton motive force (pmf). The use of these factors and forces is probably primarily determined by specific structural features of an IMP. To analyze these features we have engineered a set of model IMPs based on endogenous E. coli IMPs known to follow distinct targeting and insertion pathways. The modified model IMPs were analyzed for altered routing using an in vivo protease mapping approach. The data suggest a facultative use of different combinations of factors.     

Multiscale simulation of protein mediated membrane remodeling

Proteins interacting with membranes can result in substantial membrane deformations and curvatures. This effect is known in its broadest terms as membrane remodeling. This review article will survey current multiscale simulation methodologies that have been employed to examine protein mediated membrane remodeling.     

Acinetobacter baumannii outer membrane protein A modulates the biogenesis of outer membrane vesicles

Acinetobacter baumannii secretes outer membrane vesicles (OMVs) during both in vitro and in vivo growth, but the biogenesis mechanism by which A. baumannii produces OMVs remains undefined. Outer membrane protein A of A. baumannii (AbOmpA) is a major protein in the outer membrane and the C-terminus of AbOmpA interacts with diaminopimelate of peptidoglycan. This study investigated the role of AbOmpA in the biogenesis of A. baumannii OMVs. Quantitative and qualitative approaches were used to analyze OMV biogenesis in A. baumannii ATCC 19606T and an isogenic ΔAbOmpA mutant. OMV production was significantly increased in the ΔAbOmpA mutant compared to wild-type bacteria as demonstrated by quantitation of proteins and lipopolysaccharides (LPS) packaged in OMVs. LPS profiles prepared from OMVs from wild-type bacteria and the ΔAbOmpA mutant had identical patterns, but proteomic analysis showed different protein constituents in OMVs from wild-type bacteria compared to the ΔAbOmpA mutant. In conclusion, AbOmpA influences OMV biogenesis by controlling OMV production and protein composition.     

Cracking outer membrane biogenesis

The outer membrane is a distinguishing feature of the Gram-negative envelope. It lies on the external face of the peptidoglycan sacculus and forms a robust permeability barrier that protects extracytoplasmic structures from environmental insults. Overcoming the barrier imposed by the outer membrane presents a significant hurdle towards developing novel antibiotics that are effective against Gram-negative bacteria. As the outer membrane is an essential component of the cell, proteins involved in its biogenesis are themselves promising antibiotic targets. Here, we summarize key findings that have built our understanding of the outer membrane. Foundational studies describing the discovery and composition of the outer membrane as well as the pathways involved in its construction are discussed.     

Dual role of mitofilin in mitochondrial membrane organization and protein biogenesis

The mitochondrial inner membrane consists of two domains, inner boundary membrane and cristae membrane that are connected by crista junctions. Mitofilin/Fcj1 was reported to be involved in formation of crista junctions, however, different views exist on its function and possible partner proteins. We report that mitofilin plays a dual role. Mitofilin is part of a large inner membrane complex, and we identify five partner proteins as constituents of the mitochondrial inner membrane organizing system (MINOS) that is required for keeping cristae membranes connected to the inner boundary membrane. Additionally, mitofilin is coupled to the outer membrane and promotes protein import via the mitochondrial intermembrane space assembly pathway. Our findings indicate that mitofilin is a central component?of MINOS and functions as a multifunctional regulator of mitochondrial architecture and protein biogenesis.     

The mitochondrial import protein Mim1 promotes biogenesis of multispanning outer membrane proteins

The mitochondrial outer membrane contains translocase complexes for the import of precursor proteins. The translocase of the outer membrane complex functions as a general preprotein entry gate, whereas the sorting and assembly machinery complex mediates membrane insertion of β-barrel proteins of the outer membrane. Several α-helical outer membrane proteins are known to carry multiple transmembrane segments; however, only limited information is available on the biogenesis of these proteins. We report that mitochondria lacking the mitochondrial import protein 1 (Mim1) are impaired in the biogenesis of multispanning outer membrane proteins, whereas overexpression of Mim1 stimulates their import. The Mim1 complex cooperates with the receptor Tom70 in binding of precursor proteins and promotes their insertion and assembly into the outer membrane. We conclude that the Mim1 complex plays a central role in the import of α-helical outer membrane proteins with multiple transmembrane segments.     

The bacterial Sec-translocase: structure and mechanism

Most bacterial secretory proteins pass across the cytoplasmic membrane via the translocase, which consists of a protein-conducting channel SecYEG and an ATP-dependent motor protein SecA. The ancillary SecDF membrane protein complex promotes the final stages of translocation. Recent years have seen a major advance in our understanding of the structural and biochemical basis of protein translocation, and this has led to a detailed model of the translocation mechanism.     

A ribosome–nascent chain sensor of membrane protein biogenesis in Bacillus subtilis

Proteins in the YidC/Oxa1/Alb3 family have essential functions in membrane protein insertion and folding. Bacillus subtilis encodes two YidC homologs, one that is constitutively expressed (spoIIIJ/yidC1) and a second (yqjG/yidC2) that is induced in spoIIIJ mutants. Regulated induction of yidC2 allows B. subtilis to maintain capacity of the membrane protein insertion pathway. We here show that a gene located upstream of yidC2 (mifM/yqzJ) serves as a sensor of SpoIIIJ activity that regulates yidC2 translation. Decreased SpoIIIJ levels or deletion of the MifM transmembrane domain arrests mifM translation and unfolds an mRNA hairpin that otherwise blocks initiation of yidC2 translation. This regulated translational arrest and yidC2 induction require a specific interaction between the MifM C‐terminus and the ribosomal polypeptide exit tunnel. MifM therefore acts as a ribosome–nascent chain complex rather than as a fully synthesized protein. B. subtilis MifM and the previously described secretion monitor SecM in Escherichia coli thereby provide examples of the parallel evolution of two regulatory nascent chains that monitor different protein export pathways by a shared molecular mechanism.     

Vacuole membrane protein 1 is an endoplasmic reticulum protein required for organelle biogenesis, protein secretion, and development

Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1(-) Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1(-) cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised.     

Structure,function, and biogenesis of SecY,an integral membrane protein involved in protein export

TheE. coli secY (prlA) gene, located in the operator-distal part of thespec ribosomal protein operon, codes for an integral membrane protein, SecY. The phenotypes of temperature-sensitive and cold-sensitive mutations insecY suggest that the SecY protein plays an essential rolein vivo to facilitate protein translocation, whereas theprlA mutations in this gene suggest that SecY may interact with the signal sequence of translocating polypeptides. SecY contains most probably six cytoplasmic and five periplasmic domains, as well as 10 transmembrane segments. Such membrane-embedded structure may confer the SecY protein a translocator function, in which it provides a proteinaceous pathway for passage of secreted as well as membrane proteins. Results obtained byin vitro analyses of the translocation reactions, as well as some new phenotypes of thesecY mutants, are consistent with this notion. Possible interaction of SecY with other secretion and chaperone-like factors is also discussed.     

Mammalian cell mutants of membrane phospholipid biogenesis

Cultured mammalian cell mutants defective in the biosynthesis of membrane phospholipids, although limited in number, are increasing our understanding of the molecular mechanisms underlying the biogenesis and the biological significance of membrane phospholipids in higher eukaryotes. This review summarizes the progress in the isolation and characterization of such mutants, focusing on those isolated from cultured Chinese hamster ovary (CHO) cells.     

Toward unraveling membrane biogenesis in mammalian autophagy

Autophagy is a unique form of membrane trafficking, which delivers macromolecules and organelles from the cytoplasm to lysosomes for degradation. This fundamental and ubiquitous process in eukaryotic cells is mediated by the double-membrane-bound structures called autophagosomes, which transiently emerge in the cytoplasm. The recent remarkable explosion of knowledge of autophagy has revealed its multiple roles, especially in mammals; in addition to its basic role in turnover and reuse of cellular constituents, the process unexpectedly functions in elimination of invading bacteria and antigen presentation. Analysis of mammalian homologs of the autophagy-related (Atg) proteins identified in yeast has shed light on not only the common molecular mechanisms underlying autophagosome formation, but also specialized mechanisms that are related to the diverse functions and complex regulation of autophagy in higher organisms.