How do BCL-2 proteins induce mitochondrial outer membrane permeabilization?

The mitochondrial pathway of apoptosis proceeds when molecules sequestered between the outer and inner mitochondrial membranes are released to the cytosol by mitochondrial outer membrane permeabilization (MOMP). This process is controlled by the BCL-2 family, which is composed of both pro- and anti-apoptotic proteins. Although there is no disagreement that BCL-2 proteins regulate apoptosis, the mechanism leading to MOMP remains controversial. Current debate focuses on what interactions within the family are crucial to initiate MOMP. Specifically, do the BH3-only proteins directly engage BAX and/or BAK activation or do these proteins solely promote apoptosis by neutralization of anti-apoptotic BCL-2 proteins? We describe these models and contend that BH3-only proteins must perform both functions to efficiently engage MOMP and apoptosis.     

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 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.     

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.     

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.     

TIM23-mediated insertion of transmembrane α-helices into the mitochondrial inner membrane

While overall hydrophobicity is generally recognized as the main characteristic of transmembrane (TM) α-helices, the only membrane system for which there are detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells. Here, we provide comparable data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the TM segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated difference between the two insertion systems.     

Phospholipid composition of highly purified mitochondrial outer membranes of rat liver and Neurospora crassa. Is cardiolipin present in the mitochondrial outer membrane?

Isolated mitochondrial outer membrane vesicles (OMV) are a suitable system for studying various functions of the mitochondrial outer membrane. For studies on mitochondrial lipid import as well as for studies on the role of lipids in processes occurring in the outer membrane, knowledge of the phospholipid composition of the outer membrane is indispensable. Recently, a mild subfractionation procedure was described for the isolation of highly purified OMV from mitochondria of Neurospora crassa (Mayer, A., Lill, R. and Neupert, W. (1993) J. Cell Biol. 121, 1233–1243). This procedure, which consists of swelling and mechanical disruption of mitochondria followed by two steps of sucrose density gradient centrifugation, was adapted for the isolation of OMV from rat liver mitochondria. Using the appropriate enzyme markers it is shown that the resulting OMV are obtained in a yield of 25%, and that their purity is superior to that of previous OMV preparations. Analysis of the phospholipid composition of the OMV showed that phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol are the major phospholipid constituents, and that cardiolipin is only present in trace amounts. The phospholipid composition is very similar to that of the highly purified OMV from mitochondria of Neurospora crassa, although the latter still contain a small amount of cardiolipin.     

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 does sucrose stabilize the native state of globular proteins?

It is well known that sucrose stabilizes the native state of globular proteins against both chemical denaturants and temperature. A largely accepted explanation of sucrose-induced stabilization is not yet emerged. It is shown that the same theoretical approach able to rationalize the occurrence of cold denaturation, the contrasting role of GdmCl and Gdm₂SO₄, and the TMAO counteraction of urea denaturing activity [PCCP 12 (2010) 14245; PCCP 13 (2011) 12008; PCCP 13 (2011) 17689] works well also in the case of sucrose. The solvent-excluded volume effect plays the fundamental role because sucrose addition to water causes a marked increase in volume packing density due to the large size of sucrose molecules, that act as crowding agents.     

Biogenesis of mitochondrial β-barrel proteins: the POTRA domain is involved in precursor release from the SAM complex

The mitochondrial outer membrane contains proteinaceous machineries for the translocation of precursor proteins. The sorting and assembly machinery (SAM) is required for the insertion of β-barrel proteins into the outer membrane. Sam50 is the channel-forming core subunit of the SAM complex and belongs to the BamA/Sam50/Toc75 family of proteins that have been conserved from Gram-negative bacteria to mitochondria and chloroplasts. These proteins contain one or more N-terminal polypeptide transport-associated (POTRA) domains. POTRA domains can bind precursor proteins, however, different views exist on the role of POTRA domains in the biogenesis of β-barrel proteins. It has been suggested that the single POTRA domain of mitochondrial Sam50 plays a receptor-like function at the SAM complex. We established a system to monitor the interaction of chemical amounts of β-barrel precursor proteins with the SAM complex of wild-type and mutant yeast in organello. We report that the SAM complex lacking the POTRA domain of Sam50 efficiently binds β-barrel precursors, but is impaired in the release of the precursors. These results indicate the POTRA domain of Sam50 is not essential for recognition of β-barrel precursors but functions in a subsequent step to promote the release of precursor proteins from the SAM complex.     

Assembly of β-barrel proteins into bacterial outer membranes

Membrane proteins with a β-barrel topology are found in the outer membranes of Gram-negative bacteria and in the plastids and mitochondria of eukaryotic cells. The assembly of these membrane proteins depends on a protein folding reaction (to create the barrel) and an insertion reaction (to integrate the barrel within the outer membrane). Experimental approaches using biophysics and biochemistry are detailing the steps in the assembly pathway, while genetics and bioinformatics have revealed a sophisticated production line of cellular components that catalyze the assembly pathway in vivo. This includes the modular BAM complex, several molecular chaperones and the translocation and assembly module (the TAM). Recent screens also suggest that further components of the pathway might remain to be discovered. We review what is known about the process of β-barrel protein assembly into membranes, and the components of the β-barrel assembly machinery. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Incorporation of β-sitosterol into mitochondrial membrane enhances mitochondrial function by promoting inner mitochondrial membrane fluidity

Recent findings suggest that mitochondrial membrane fluidity could influence mitochondrial energy metabolism. β-sitosterol (BS) is a common plant sterol that is prevalent in plant oils, nuts, cereals and plant food products. Its chemical structure is very similar to that of cholesterol. As a cholesterol analog, BS is highly lipid soluble and largely resides in the membranes of cells or organelles where it may have an influence on the membrane fluidity. The present study reports that, with the cholesterol chelator 2-hydroxypropyl-β-cyclodextrin (HPβCD) as its carrier, BS is able to increase the fluidity of the inner mitochondrial membrane (IMM) without affecting the fluidity of the outer mitochondrial membrane (OMM), and consequently to increase the mitochondrial membrane potential (?Ψm) and mitochondrial ATP content. It has been previously proposed that a therapeutical boost in adenosine triphosphate (ATP) levels in mitochondria may be beneficial for neurodegenerative diseases such as Alzheimer’s disease (AD). Given that dietary administration of plant sterols could increase brain BS concentrations, these results may provide a better understanding of the beneficial effects of plant sterol-enriched nutrients on neurodegenerative diseases such as AD.     

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 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.