How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane?

Bcl-2 family members, like the structurally similar translocation domain of diphtheria toxin, can form ion-selective channels and larger-diameter pores in artificial lipid bilayers. Recent studies show how Bcl-2 family members change topology in membranes during apoptosis and that these different states may either promote or inhibit apoptosis. Binding of BH3-only proteins alters the subcellular localization and/or membrane topology and probably affects the channel formation of Bcl-2, Bcl-xL and Bcl-w. However, it remains unclear how the pore-forming activity functions in cells to regulate mitochondrial membrane permeabilization and cell death. Bcl-2 family members in flies and worms regulate apoptosis by mechanisms seemingly unrelated to membrane permeabilization, leaving a unifying model for the biochemical activity of this protein family unknown. Work linking Bcl-2 family members to mitochondrial morphogenesis in worms and mammals suggests some common functions of Bcl-2 family proteins may exist.     

How do membrane proteins sense water stress?

Maintenance of cell turgor is a prerequisite for almost any form of life as it provides a mechanical force for the expansion of the cell envelope. As changes in extracellular osmolality will have similar physicochemical effects on cells from all biological kingdoms, the responses to osmotic stress may be alike in all organisms. The primary response of bacteria to osmotic upshifts involves the activation of transporters, to effect the rapid accumulation of osmoprotectants, and sensor kinases, to increase the transport and/or biosynthetic capacity for these solutes. Upon osmotic downshift, the excess of cytoplasmic solutes is released via mechanosensitive channel proteins. A number of breakthroughs in the last one or two years have led to tremendous advances in our understanding of the molecular mechanisms of osmosensing in bacteria. The possible mechanisms of osmosensing, and the actual evidence for a particular mechanism, are presented for well studied, osmoregulated transport systems, sensor kinases and mechanosensitive channel proteins. The emerging picture is that intracellular ionic solutes (or ionic strength) serve as a signal for the activation of the upshift-activated transporters and sensor kinases. For at least one system, there is strong evidence that the signal is transduced to the protein complex via alterations in the protein-lipid interactions rather than direct sensing of ion concentration or ionic strength by the proteins. The osmotic downshift-activated mechanosensitive channels, on the other hand, sense tension in the membrane but other factors such as hydration state of the protein may affect the equilibrium between open and closed states of the proteins.     

How does the TOM complex mediate insertion of precursor proteins into the mitochondrial outer membrane?

A multisubunit translocase of the outer mitochondrial membrane (TOM complex) mediates both the import of mitochondrial precursor proteins into the internal compartments of the organelle and the insertion of proteins residing in the mitochondrial outer membrane. The proposed beta-barrel structure of Tom40, the pore-forming component of the translocase, raises the question of how the apparent uninterrupted beta-barrel topology can be compatible with a role of Tom40 in releasing membrane proteins into the lipid core of the bilayer. In this review, I discuss insertion mechanisms of proteins into the outer membrane and present alternative models based on the opening of a multisubunit beta-barrel TOM structure or on the interaction of outer membrane precursors with the outer face of the Tom40 beta-barrel structure.     

How do Rab proteins function in membrane traffic?

The Rabs are a group of GTP-binding proteins implicated for some time in the targeting of different transport vesicles within the cell, but it has been unclear how they function, or how they relate to a second group of targeting proteins, the SNAREs. Recent work, discussed in this review, has used biophysical, biochemical and genetic approaches to begin to answer these questions for Rab3, Rab5 and the yeast protein Sec4p. However, the results from these three Rabs lead to a surprising conclusion: the different Rabs seem to function via highly diverse target proteins.     

Structure and evolution of mitochondrial outer membrane proteins of β-barrel topology

Gram-negative bacteria are the ancestors of mitochondrial organelles. Consequently, both entities contain two surrounding lipid bilayers known as the inner and outer membranes. While protein synthesis in bacteria is accomplished in the cytoplasm, mitochondria import 90–99% of their protein ensemble from the cytosol in the opposite direction. Three protein families including Sam50, VDAC and Tom40 together with Mdm10 compose the set of integral β-barrel proteins embedded in the mitochondrial outer membrane in S. cerevisiae (MOM). The 16-stranded Sam50 protein forms part of the sorting and assembly machinery (SAM) and shows a clear evolutionary relationship to members of the bacterial Omp85 family. By contrast, the evolution of VDAC and Tom40, both adopting the same fold cannot be traced to any bacterial precursor. This finding is in agreement with the specific function of Tom40 in the TOM complex not existent in the enslaved bacterial precursor cell. Models of Tom40 and Sam50 have been developed using X-ray structures of related proteins. These models are analyzed with respect to properties such as conservation and charge distribution yielding features related to their individual functions.     

How do proteins fold?

This article emphasizes the importance of getting students to understand the ways in which polypeptides fold to form protein molecules with complex higher-ordered structures. Modern views on how this folding occurs in vitro and in the cell are summarized and set within an appropriate biological context.     

Characterization of the insertase for β-barrel proteins of the outer mitochondrial membrane

The TOB–SAM complex is an essential component of the mitochondrial outer membrane that mediates the insertion of β-barrel precursor proteins into the membrane. We report here its isolation and determine its size, composition, and structural organization. The complex from Neurospora crassa was composed of Tob55–Sam50, Tob38–Sam35, and Tob37–Sam37 in a stoichiometry of 1:1:1 and had a molecular mass of 140 kD. A very minor fraction of the purified complex was associated with one Mdm10 protein. Using molecular homology modeling for Tob55 and cryoelectron microscopy reconstructions of the TOB complex, we present a model of the TOB–SAM complex that integrates biochemical and structural data. We discuss our results and the structural model in the context of a possible mechanism of the TOB insertase.     

How many membrane proteins are there?

One of the basic issues that arises in functional genomics is the ability to predict the subcellular location of proteins that are deduced from gene and genome sequencing. In particular, one would like to be able to readily specify those proteins that are soluble and those that are inserted in a membrane. Traditional methods of distinguishing between these two locations have relied on extensive, time-consuming biochemical studies. The alternative approach has been to make inferences based on a visual search of the amino acid sequences of presumed gene products for stretches of hydrophobic amino acids. This numerical, sequence-based approach is usually seen as a first approximation pending more reliable biochemical data. The recent availability of large and complete sequence data sets for several organisms allows us to determine just how accurate such a numerical approach could be, and to attempt to minimize and quantify the error involved. We have optimized a statistical approach to protein location determination. Using our approach, we have determined that surprisingly few proteins are misallocated using the numerical method. We also examine the biological implications of the success of this technique.     

How do uncoupling proteins uncouple?

According to the proton buffering model, introduced by Klingenberg, UCP1 conducts protons through a hydrophilic pathway lined with fatty acid head groups that buffer the protons as they move across the membrane. According to the fatty acid protonophore model, introduced by Garlid, UCPs do not conduct protons at all. Rather, like all members of this gene family, they are anion carriers. A variety of anions are transported, but the physiological substrates are fatty acid (FA) anions. Because the carboxylate head group is translocated by UCP, and because the protonated FA rapidly diffuses across the membrane, this mechanism permits FA to behave as regulated cycling protonophores. Favoring the latter mechanism is the fact that the head group of long-chain alkylsulfonates, strong acid analogues of FA, is also translocated by UCP.     

How do mangrove forests induce sedimentation?

The mangrove forests play a significant role as sediment traps. They reduce tidal flows and induce sedimentation of soil particles at low tide. However, there are no such processes taking place in the non-mangrove areas. Site of Avicennia-Rhizophora interphase is more efficient than Avicennia and or Rhizophora zones, in trapping the sediment by 30, 25 and 20% respectively at low tide as compared to high tide.     

How do thermophilic proteins resist aggregation?

Aggregation is an ancient threat that must be overcome by proteins from all organisms to maintain their native functional states. This is essential for the maintenance of metabolic flux and viability of their cellular machineries. Here, we compare the aggregation-resistance strategies adapted by the thermophilic proteins and their mesophilic homologs using a dataset of 373 protein families. Like their mesophilic homologs, the thermophilic protein sequences also contain potential aggregation prone regions (APRs), capable of forming cross-β motif and amyloid-like fibrils. Tetrapeptide and hexapeptide amyloid-like fibril forming sequence patterns and experimentally proven amyloid-like fibril forming peptide sequences were also detected in the thermophilic proteins. Both the thermophilic and the mesophilic proteins use similar strategies to resist aggregation. However, the thermophilic proteins show superior utilization of these strategies. The thermophilic protein monomers show greater ability to "stow away" the APRs in the hydrophobic cores to protect them from solvent exposure. The thermophilic proteins are also better at gatekeeping the APRs by surrounding them with charged residues (Asp, Glu, Lys, and Arg) and Pro to a greater extent. While thermophilic and mesophilic proteins in our dataset are highly homologous and show strong overall sequence conservation, the APRs are not conserved between the homologs. These findings indicate that evolution is working to avoid amyloidogenic regions in proteins. Our results are also consistent with the observation that thermophilic cells often accumulate small molecule osmolytes capable of stabilizing their proteins and other macromolecules. This study has important implications for rational design and formulation of therapeutic proteins and antibodies.     

How does lysozyme penetrate through the bacterial outer membrane?

Lysozyme fails to penetrate through the outer membrane of stationary phase cells of Escherichia coli when it is simply added to suspensions of plasmolyzed cells. Lysozyme penetrates the outer membrane only when these cells are exposed to a mild osmotic shock in the presence of EDTA and lysozyme.In the presence of Mg²⁺, the outer membrane is stabilized sufficiently so that there is no lysozyme penetration during osmotic shock. If Mg²⁺ is added after an osmotic shock has been used to cause lysozyme to penetrate a destabilized outer membrane, the outer membrane is stabilized once again. In this case however, cells are converted to spheroplasts by the lysozyme which has gained access to the murein layer prior to the addition of Mg²⁺. Mg²⁺ stabilizes the outer membranes of these spheroplasts sufficiently so that they remain immune to lysis even in the absence of osmotic stabilizers such as sucrose.These results are discussed in terms of current information on the structure of the murein layer and the outer membrane.     

Wza: a new structural paradigm for outer membrane secretory proteins?

Gram-negative bacteria need to be able to transport a large variety of macromolecules across their outer membranes. In Escherichia coli, the passage of the group 1 capsular polysaccharide is mediated by an integral outer membrane protein, Wza. The crystal structure of Wza, determined recently, reveals a novel transmembrane alpha-helical barrel and a large central cavity within the core of the vase-shaped protein complex. The structure has similarities with that of the secretin protein, PilQ, which mediates the transition of type IV pili across the outer membrane. We propose that the large internal chamber, which can accommodate the secreted assembled macromolecule, is likely to be a common feature found in other outer membrane proteins involved in secretion processes.     

Biogenesis of β-barrel integral proteins of bacterial outer membrane

Gram-negative bacteria are enveloped by two membranes, the inner (cytoplasmic) (CM) and the outer (OM). The majority of integral outer membrane proteins are arranged in β-barrels of cylindrical shape composed of amphipathic antiparallel β-strands. In bacteria, β-barrel proteins function as water-filled pores, active transporters, enzymes, receptors, and structural proteins. Proteins of bacterial OM are synthesized in the cytoplasm as unfolded polypeptides with an N-terminal sequence that marks them for transport across the CM. Precursors of membrane proteins move through the aqueous medium of the cytosol and periplasm under the protection of chaperones (SecB, Skp, SurA, and DegP), then cross the CM via the Sec system composed of a polypeptide-conducting channel (SecYEG) and ATPase (SecA), the latter providing the energy for the translocation of the pre-protein. Pre-protein folding and incorporation in the OM require the participation of the Bam-complex, probably without the use of energy. This review summarizes current data on the biogenesis of the β-barrel proteins of bacterial OM. Data on the structure of the proteins included in the multicomponent system for delivery of the OM proteins to their destination in the cell and on their complexes with partners, including pre-proteins, are pre-sented. Molecular models constructed on the basis of structural, genetic, and biochemical studies that describe the mechanisms of β-barrel protein assembly by this molecular transport machinery are also considered.     

How important are transmembrane helices of bitopic membrane proteins?

The topology of a bitopic membrane protein consists of a single transmembrane helix connecting two extra-membranous domains. As opposed to helices from polytopic proteins, the transmembrane helices of bitopic proteins were initially considered as merely hydrophobic anchors, while more recent studies have begun to shed light on their role in the protein's function. Herein the overall importance of transmembrane helices from bitopic membrane proteins was analyzed using a relative conservation analysis. Interestingly, the transmembrane domains of bitopic proteins are on average, significantly more conserved than the remainder of the protein, even when taking into account their smaller amino acid repertoire. Analysis of highly conserved transmembrane domains did not reveal any unifying consensus, pointing to a great diversity in their conservation patterns. However, Fourier power spectrum analysis was able to show that regardless of the conservation motif, in most sequences a significant conservation moment was observed, in that one side of the helix was conserved while the other was not. Taken together, it may be possible to conclude that a significant proportion of transmembrane helices from bitopic membrane proteins participate in specific interactions, in a variety of modes in the plane of the lipid bilayer.