Mapping of the protein import machinery in the mitochondrial outer membrane by crosslinking of translocation intermediates.

Mitochondria contain a complex machinery for the import of nuclear-encoded proteins. Receptor proteins exposed on the outer membrane surface are required for the specific binding of precursor proteins to mitochondria, either by binding of cytosolic signal recognition factors or by direct recognition of the precursor polypeptides. Subsequently, the precursors are inserted into the outer membrane at the general insertion site GIP (general insertion protein). Here we report the analysis of receptors and GIP by crosslinking of translocation intermediates and by coimmunoprecipitation. Surface-accumulated precursors were crosslinked to the receptors MOM19 and MOM72, suggesting a direct interaction of preproteins with surface receptors. We identified three novel mitochondrial outer membrane proteins, MOM7, MOM8, and MOM30 that, together with the previously identified MOM38, seem to form the GIP site and are present in the mitochondrial receptor complex.     

Evolutionary conservation of biogenesis of beta-barrel membrane proteins

The outer membranes of mitochondria and chloroplasts are distinguished by the presence of beta-barrel membrane proteins. The outer membrane of Gram-negative bacteria also harbours beta-barrel proteins. In mitochondria these proteins fulfil a variety of functions such as transport of small molecules (porin/VDAC), translocation of proteins (Tom40) and regulation of mitochondrial morphology (Mdm10). These proteins are encoded by the nucleus, synthesized in the cytosol, targeted to mitochondria as chaperone-bound species, recognized by the translocase of the outer membrane, and then inserted into the outer membrane where they assemble into functional oligomers. Whereas some knowledge has been accumulated on the pathways of insertion of proteins that span cellular membranes with alpha-helical segments, very little is known about how beta-barrel proteins are integrated into lipid bilayers and assembled into oligomeric structures. Here we describe a protein complex that is essential for the topogenesis of mitochondrial outer membrane beta-barrel proteins (TOB). We present evidence that important elements of the topogenesis of beta-barrel membrane proteins have been conserved during the evolution of mitochondria from endosymbiotic bacterial ancestors.     

Machinery for protein sorting and assembly in the mitochondrial outer membrane

Mitochondria contain translocases for the transport of precursor proteins across their outer and inner membranes. It has been assumed that the translocases also mediate the sorting of proteins to their submitochondrial destination. Here we show that the mitochondrial outer membrane contains a separate sorting and assembly machinery (SAM) that operates after the translocase of the outer membrane (TOM). Mas37 forms a constituent of the SAM complex. The central role of the SAM complex in the sorting and assembly pathway of outer membrane proteins explains the various pleiotropic functions that have been ascribed to Mas37 (refs 4, 11-15). These results suggest that the TOM complex, which can transport all kinds of mitochondrial precursor proteins, is not sufficient for the correct integration of outer membrane proteins with a complicated topology, and instead transfers precursor proteins to the SAM complex.     

The mechanism of membrane-associated steps in tail-anchored protein insertion

Tail-anchored (TA) membrane proteins destined for the endoplasmic reticulum are chaperoned by cytosolic targeting factors that deliver them to a membrane receptor for insertion. Although a basic framework for TA protein recognition is now emerging, the decisive targeting and membrane insertion steps are not understood. Here we reconstitute the TA protein insertion cycle with purified components, present crystal structures of key complexes between these components and perform mutational analyses based on the structures. We show that a committed targeting complex, formed by a TA protein bound to the chaperone ATPase Get3, is initially recruited to the membrane through an interaction with Get2. Once the targeting complex has been recruited, Get1 interacts with Get3 to drive TA protein release in an ATPase-dependent reaction. After releasing its TA protein cargo, the now-vacant Get3 recycles back to the cytosol concomitant with ATP binding. This work provides a detailed structural and mechanistic framework for the minimal TA protein insertion cycle.     

YidC mediates membrane protein insertion in bacteria

The basic machinery for the translocation of proteins into or across membranes is remarkably conserved from Escherichia coli to humans. In eukaryotes, proteins are inserted into the endoplasmic reticulum using the signal recognition particle (SRP) and the SRP receptor, as well as the integral membrane Sec61 trimeric complex (composed of alpha, beta and gamma subunits). In bacteria, most proteins are inserted by a related pathway that includes the SRP homologue Ffh, the SRP receptor FtsY, and the SecYEG trimeric complex, where Y and E are related to the Sec61 alpha and gamma subunits, respectively. Proteins in bacteria that exhibit no dependence on the Sec translocase were previously thought to insert into the membrane directly without the aid of a protein machinery. Here we show that membrane insertion of two Sec-independent proteins requires YidC. YidC is essential for E. coli viability and homologues are present in mitochondria and chloroplasts. Depletion of YidC also interferes with insertion of Sec-dependent membrane proteins, but it has only a minor effect on the export of secretory proteins. These results provide evidence for an additional component of the translocation machinery that is specialized for the integration of membrane proteins.     

Recognition of transmembrane helices by the endoplasmic reticulum translocon

Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a 'biological' hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein-lipid interactions are critical during translocon-mediated membrane insertion.     

Protein targeting and degradation are coupled for elimination of mislocalized proteins

A substantial proportion of the genome encodes membrane proteins that are delivered to the endoplasmic reticulum by dedicated targeting pathways. Membrane proteins that fail targeting must be rapidly degraded to avoid aggregation and disruption of cytosolic protein homeostasis. The mechanisms of mislocalized protein (MLP) degradation are unknown. Here we reconstitute MLP degradation in vitro to identify factors involved in this pathway. We find that nascent membrane proteins tethered to ribosomes are not substrates for ubiquitination unless they are released into the cytosol. Their inappropriate release results in capture by the Bag6 complex, a recently identified ribosome-associating chaperone. Bag6-complex-mediated capture depends on the presence of unprocessed or non-inserted hydrophobic domains that distinguish MLPs from potential cytosolic proteins. A subset of these Bag6 complex 'clients' are transferred to TRC40 for insertion into the membrane, whereas the remainder are rapidly ubiquitinated. Depletion of the Bag6 complex selectively impairs the efficient ubiquitination of MLPs. Thus, by its presence on ribosomes that are synthesizing nascent membrane proteins, the Bag6 complex links targeting and ubiquitination pathways. We propose that such coupling allows the fast tracking of MLPs for degradation without futile engagement of the cytosolic folding machinery.     

Calpain的激活与鱼藤酮导致的细胞凋亡

在缺氧或呼吸链抑制剂存在条件下,细胞的呼吸链受到抑制,线粒体的功能受到直接干扰,细胞色素C通过线粒体的外膜特异性通道进入细胞浆内,启动了procaspase-3等一系列凋亡因子,细胞发生与线粒体相关的凋亡。另一方面,因线粒体的功能被抑制,细胞内的钙离子浓度升高,calpain被激活并裂解细胞膜蛋白及细胞内的生物化学分子,促进了细胞凋亡的发生。鱼藤酮作为线粒体呼吸链complexI的抑制剂可导致细胞凋亡,其凋亡途径不仅与caspase介导的机制有关,还有可能与calpain有关。     

Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching

Mitochondrial uncoupling protein 2 (UCP2) is an integral membrane protein in the mitochondrial anion carrier protein family, the members of which facilitate the transport of small molecules across the mitochondrial inner membrane. When the mitochondrial respiratory complex pumps protons from the mitochondrial matrix to the intermembrane space, it builds up an electrochemical potential. A fraction of this electrochemical potential is dissipated as heat, in a process involving leakage of protons back to the matrix. This leakage, or 'uncoupling' of the proton electrochemical potential, is mediated primarily by uncoupling proteins. However, the mechanism of UCP-mediated proton translocation across the lipid bilayer is unknown. Here we describe a solution-NMR method for structural characterization of UCP2. The method, which overcomes some of the challenges associated with membrane-protein structure determination, combines orientation restraints derived from NMR residual dipolar couplings (RDCs) and semiquantitative distance restraints from paramagnetic relaxation enhancement (PRE) measurements. The local and secondary structures of the protein were determined by piecing together molecular fragments from the Protein Data Bank that best fit experimental RDCs from samples weakly aligned in a DNA nanotube liquid crystal. The RDCs also determine the relative orientation of the secondary structural segments, and the PRE restraints provide their spatial arrangement in the tertiary fold. UCP2 closely resembles the bovine ADP/ATP carrier (the only carrier protein of known structure), but the relative orientations of the helical segments are different, resulting in a wider opening on the matrix side of the inner membrane. Moreover, the nitroxide-labelled GDP binds inside the channel and seems to be closer to transmembrane helices 1-4. We believe that this biophysical approach can be applied to other membrane proteins and, in particular, to other mitochondrial carriers, not only for structure determination but also to characterize various conformational states of these proteins linked to substrate transport.     

Rapid regulation of steroidogenesis by mitochondrial protein import

Most mitochondrial proteins are synthesized on cytoplasmic ribosomes and imported into mitochondria. The imported proteins are directed to one of four submitochondrial compartments--the outer mitochondrial membrane, the inner mitochondrial membrane, the intramembraneous space, or the matrix--where the protein then functions. Here we show that the steroidogenic acute regulatory protein (StAR), a mitochondrial protein required for stress responses, reproduction, and sexual differentiation of male fetuses, exerts its activity transiently at the outer mitochondrial membrane rather than at its final resting place in the matrix. We also show that its residence time at this outer membrane and its activity are regulated by its speed of mitochondrial import. This may be the first example of a mitochondrial protein exerting its biological activity in a compartment other than that to which it is finally targeted. This system enables steroidogenic cells to initiate and terminate massive levels of steroidogenesis within a few minutes, permitting the rapid regulation of serum steroid hormone concentrations.     

A family of mitochondrial proteins involved in bioenergetICS and biogenesis

The respiratory chain complexes of mitochondria consist of many different subunits, of which only a few partake directly in electron transport. The functions of the subunits that do not contain prosthetic groups are largely unknown. The cytochrome reductase complex of Neurospora crassa, for examine, consists of nine different subunits, of which the peripheral membrane proteins I and II (ref.3) that are located on the matrix side of the mitochondrial inner membrane are the largest subunits devoid of redox centres. Significantly, a cytochrome reductase fraction lacking these two subunits was inactive in electron transfer, and in yeast mutants with defective genes for either of the two subunits, assembly of the reductase is disrupted. Most mitochondrial proteins are imported into the mitochondrion as precursor proteins, and two proteins are necessary for cleaving their presequences, namely the matrix processing peptidase (MPP) and the processing enhancing protein (PEP), the latter strongly stimulating the activity of the former. Temperature-sensitive yeast mutants, which are affected in PEP or MPP, accumulate precursors at the nonpermissive temperature. We report here that subunit I of the cytochrome reductase can be grouped as members of the same protein family.     

A 'molten-globule' membrane-insertion intermediate of the pore-forming domain of colicin A

The 'molten' globular conformation of a protein is compact with a native secondary structure but a poorly defined tertiary structure. Molten globular states are intermediates in protein folding and unfolding and they may be involved in the translocation or insertion of proteins into membranes. Here we investigate the membrane insertion of the pore-forming domain of colicin A, a bacteriocin that depolarizes the cytoplasmic membrane of sensitive cells. We find that this pore-forming domain, the insertion of which depends on pH, undergoes a native to molten globule transition at acidic pH. The variation of the kinetic constant of membrane insertion of the protein into negatively charged lipid vesicles as a function of the interfacial pH correlates with the appearance of the acidic molten globular state, indicating that this state could be an intermediate formed during the insertion of colicin A into membranes.     

Isolation and characterization of the gene for a yeast mitochondrial import receptor

We have previously identified an integral membrane protein (p32) from Saccharomyces cerevisiae as a receptor for protein import into mitochondria, and have localized it to the mitochondrial outer membrane at contact sites. Here we report isolation of the corresponding mitochondrial import receptor gene, termed MIR1. The deduced amino-acid sequence of p32 shows roughly 40% identity with proteins of bovine heart and rat liver that have been suggested to be mitochondrial phosphate carriers. Haploid cells carrying a disrupted MIR1 allele were unable to grow on a non-fermentable carbon source but grew in media containing glucose, indicating that the MIR1 protein is essential for mitochondrial function. Compared with wild type, amounts of some mitochondrial proteins were markedly reduced in cells containing a disrupted MIR1 allele, whereas levels of others were unchanged. This indicates that yeast contains more than one pathway for protein import into mitochondria.     

Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues

Positively charged amino acids have been shown to be important elements in targeting-peptides that direct proteins into mitochondria, nuclei, and the secretory pathways of both prokaryotic and eukaryotic cells. The 'positive-inside' rule, which observes that regions of polytopic (multi-spanning) membrane proteins facing the cytoplasm are generally enriched in arginyl and lysyl residues whereas translocated regions are largely devoid of these residues, implies that the distribution of positively charged amino acids may also be a major determinant of the transmembrane topology of integral membrane proteins. If this is indeed the case, it should be possible to predictably alter the topology of a polytopic protein by site-directed insertions and/or deletions of positively charged residues in critical locations. I now describe a derivative of Escherichia coli leader peptidase, a polytopic inner-membrane protein, that switches from sec-gene-dependent membrane insertion with a Nout-Cout transmembrane topology to sec-gene-independent insertion with a Nin-Cin topology in response to the addition of four positively charged lysines to its N terminus.     

A substrate-specific inhibitor of protein translocation into the endoplasmic reticulum

The segregation of secretory and membrane proteins to the mammalian endoplasmic reticulum is mediated by remarkably diverse signal sequences that have little or no homology with each other. Despite such sequence diversity, these signals are all recognized and interpreted by a highly conserved protein-conducting channel composed of the Sec61 complex. Signal recognition by Sec61 is essential for productive insertion of the nascent polypeptide into the translocation site, channel gating and initiation of transport. Although subtle differences in these steps can be detected between different substrates, it is not known whether they can be exploited to modulate protein translocation selectively. Here we describe cotransin, a small molecule that inhibits protein translocation into the endoplasmic reticulum. Cotransin acts in a signal-sequence-discriminatory manner to prevent the stable insertion of select nascent chains into the Sec61 translocation channel. Thus, the range of substrates accommodated by the channel can be specifically and reversibly modulated by a cell-permeable small molecule that alters the interaction between signal sequences and the Sec61 complex.     

Post-translational insertion of a fragment of the glucose transporter into microsomes requires phosphoanhydride bond cleavage

Most eukaryotic secretory and membrane proteins insert co-translationally into the membrane of the rough endoplasmic reticulum (RER), and are targeted there by one or more NH2-terminal or internal signal sequences. However, little is known about the actual translocation and membrane integration processes. In particular, any energy requirements for targeting and integration have remained obscure because of the inability to uncouple the processes from concomitant protein synthesis. We recently showed that the human glucose transporter (GT), an integral membrane glycoprotein, can insert post-translationally into dog pancreatic microsomes with low but demonstrable efficiency in vitro, and that a fragment corresponding to the NH2-terminal 340 amino acids and 8 of the 12 membrane-spanning alpha-helixes of GT (GT-N) can insert with significantly greater efficiency. We report here that post-translational insertion of GT-N into pancreatic microsomes requires energy in the form of a phosphodiester bond, and suggest that co-translational insertion of proteins into the RER may also require energy independent of that used for polypeptide synthesis.     

多分辨预处理技术加速PO-MOM混合方法的研究

结合复杂物体散射特性的电磁计算问题,研究了物理光学(PO)与矩量法(MOM)相结合的混合技术,寻求PO-MOM方法减少计算时间和存储空间、提高计算精度的途径;针对矩量法在求解过程中的迭代问题,引入多分辨预处理技术,并给出算例.结果表明,在满足计算精度不变的前提下,使用PO-MOM混合方法提高了计算效率.     

Mitochondrial membrane remodelling regulated by a conserved rhomboid protease

Rhomboid proteins are intramembrane serine proteases that activate epidermal growth factor receptor (EGFR) signalling in Drosophila. Rhomboids are conserved throughout evolution, and even in eukaryotes their existence in species with no EGFRs implies that they must have additional roles. Here we report that Saccharomyces cerevisiae has two rhomboids, which we have named Rbd1p and Rbd2p. RBD1 deletion results in a respiratory defect; consistent with this, Rbd1p is localized in the inner mitochondrial membrane and mutant cells have disrupted mitochondria. We have identified two substrates of Rbd1p: cytochrome c peroxidase (Ccp1p); and a dynamin-like GTPase (Mgm1p), which is involved in mitochondrial membrane fusion. Rbd1p mutants are indistinguishable from Mgm1p mutants, indicating that Mgm1p is a key substrate of Rbd1p and explaining the rbd1Delta mitochondrial phenotype. Our data indicate that mitochondrial membrane remodelling is regulated by cleavage of Mgm1p and show that intramembrane proteolysis by rhomboids controls cellular processes other than signalling. In addition, mitochondrial rhomboids are conserved throughout eukaryotes and the mammalian homologue, PARL, rescues the yeast mutant, suggesting that these proteins represent a functionally conserved subclass of rhomboid proteases.     

Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC.

During transduction of an apoptotic (death) signal into the cell, there is an alteration in the permeability of the membranes of the cell's mitochondria, which causes the translocation of the apoptogenic protein cytochrome c into the cytoplasm, which in turn activates death-driving proteolytic proteins known as caspases. The Bcl-2 family of proteins, whose members may be anti-apoptotic or pro-apoptotic, regulates cell death by controlling this mitochondrial membrane permeability during apoptosis, but how that is achieved is unclear. Here we create liposomes that carry the mitochondrial porin channel (also called the voltage-dependent anion channel, or VDAC) to show that the recombinant pro-apoptotic proteins Bax and Bak accelerate the opening of VDAC, whereas the anti-apoptotic protein Bcl-x(L) closes VDAC by binding to it directly. Bax and Bak allow cytochrome c to pass through VDAC out of liposomes, but passage is prevented by Bcl-x(L). In agreement with this, VDAC1-deficient mitochondria from a mutant yeast did not exhibit a Bax/Bak-induced loss in membrane potential and cytochrome c release, both of which were inhibited by Bcl-x(L). Our results indicate that the Bcl-2 family of proteins bind to the VDAC in order to regulate the mitochondrial membrane potential and the release of cytochrome c during apoptosis.