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Myostatin, a key regulator of muscle mass in vertebrates, is biosynthesised as a latent precursor in muscle and is activated by sequential proteolysis of the pro-domain. To investigate the molecular mechanism by which pro-myostatin remains latent, we have determined the structure of unprocessed pro-myostatin and analysed the properties of the protein in its different forms. Crystal structures and SAXS analyses show that pro-myostatin adopts an open, V-shaped structure with a domain-swapped arrangement. The pro-mature complex, after cleavage of the furin site, has significantly reduced activity compared with the mature growth factor and persists as a stable complex that is resistant to the natural antagonist follistatin. The latency appears to be conferred by a number of distinct features that collectively stabilise the interaction of the pro-domains with the mature growth factor, enabling a regulated stepwise activation process, distinct from the prototypical pro-TGF-β1. These results provide a basis for understanding the effect of missense mutations in pro-myostatin and pave the way for the design of novel myostatin inhibitors., (© 2018 The Authors.)

Growth differentiation factor 8 (GDF8)/myostatin is a latent TGF-β family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid-cleaved GDF8 pro-complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro-complexes reveals a V-shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring-like, cross-armed conformation of latent TGF-β1. Surprisingly, Tolloid-cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen-deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain-growth factor dissociation, increased exchange in regions that correspond in pro-TGF-β1 to the α1-helix, latency lasso, and β1-strand in the prodomain and to the β6'- and β7'-strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain-growth factor interfaces and primes the growth factor for release from the prodomain., (© 2018 The Authors.)

Recognition of microbial challenges leads to enhanced immunity at both the local and systemic levels. In Arabidopsis, EFR and PEPR1/PEPR2 act as the receptor for the bacterial elongation factor EF-Tu (elf18 epitope) and for the endogenous PROPEP-derived Pep epitopes, respectively. The PEPR pathway has been described to mediate defence signalling following microbial recognition. Here we show that PROPEP2/PROPEP3 induction upon pathogen challenges is robust against jasmonate, salicylate, or ethylene dysfunction. Comparative transcriptome profiling between Pep2- and elf18-treated plants points to co-activation of otherwise antagonistic jasmonate- and salicylate-mediated immune branches as a key output of PEPR signalling. Accordingly, as well as basal defences against hemibiotrophic pathogens, systemic immunity is reduced in pepr1 pepr2 plants. Remarkably, PROPEP2/PROPEP3 induction is essentially restricted to the pathogen challenge sites during pathogen-induced systemic immunity. Localized Pep application activates genetically separable jasmonate and salicylate branches in systemic leaves without significant PROPEP2/PROPEP3 induction. Our results suggest that local PEPR activation provides a critical step in connecting local to systemic immunity by reinforcing separate defence signalling pathways.

The heterotrimeric SecYEG complex comprises a protein-conducting channel in the bacterial cytoplasmic membrane. SecYEG functions together with the motor protein SecA in preprotein translocation. Here, we have addressed the functional oligomeric state of SecYEG when actively engaged in preprotein translocation. We reconstituted functional SecYEG complexes labelled with fluorescent markers into giant unilamellar vesicles at a natively low density. Förster's resonance energy transfer and fluorescence (cross-) correlation spectroscopy with single-molecule sensitivity allowed for independent observations of the SecYEG and preprotein dynamics, as well as complex formation. In the presence of ATP and SecA up to 80% of the SecYEG complexes were loaded with a preprotein translocation intermediate. Neither the interaction with SecA nor preprotein translocation resulted in the formation of SecYEG oligomers, whereas such oligomers can be detected when enforced by crosslinking. These data imply that the SecYEG monomer is sufficient to form a functional translocon in the lipid membrane.

The TOM complex of the outer membrane of mitochondria is the entry gate for the vast majority of precursor proteins that are imported into the mitochondria. It is made up by receptors and a protein conducting channel. Although precursor proteins of all subcompartments of mitochondria use the TOM complex, it is not known whether its channel can only mediate passage across the outer membrane or also lateral release into the outer membrane. To study this, we have generated fusion proteins of GFP and Tim23 which are inserted into the inner membrane and, at the same time, are spanning either the TOM complex or are integrated into the outer membrane. Our results demonstrate that the TOM complex, depending on sequence determinants in the precursors, can act both as a protein conducting pore and as an insertase mediating lateral release into the outer membrane.

The thylakoid twin arginine protein translocation (Tat) system is thought to have a multivalent receptor complex with each cpTatC-Hcf106 pair constituting a signal peptide-binding unit. Conceptual models suggest that translocation of individual precursor proteins occurs upon assembly of a Tha4 oligomer with a precursor-occupied cpTatC-Hcf106. However, results reported here reveal that multiple precursor proteins bound to a single receptor complex can be transported together. Precursor proteins that contain one or two cysteine residues readily formed intermolecular disulphide bonds upon binding to the receptor complex, resulting in dimeric and tetrameric precursor proteins. Three lines of evidence indicate that all members of precursor oligomers were specifically bound to a receptor unit. Blue native-polyacrylamide gel electrophoresis analysis showed that oligomers were present on individual receptor complexes rather than bridging two or more receptor complexes. Upon energizing the membrane, the dimeric and tetrameric precursors were transported across the membrane with efficiencies comparable with that of monomeric precursors. These results imply a novel aspect of Tat systems, whereby multiple precursor-binding sites can act in concert to transport an interlinked oligo-precursor protein.

The glycosylphosphatidylinositol (GPI)-anchored proteoglycan Cripto binds Nodal and its type I receptor Alk4 to activate Smad2,3 transcription factors, but a role during Nodal precursor processing has not been described. We show that Cripto also binds the proprotein convertases Furin and PACE4 and localizes Nodal processing at the cell surface. When coexpressed as in early embryonic cells, Cripto and uncleaved Nodal already associated during secretion, and a Cripto-interacting region in the Nodal propeptide potentiated the effect of proteolytic maturation on Nodal signalling. Disruption of the trans-Golgi network (TGN) by brefeldin A blocked secretion, but export of Cripto and Nodal to the cell surface was not inhibited, indicating that Nodal is exposed to extracellular convertases before entering the TGN/endosomal system. Density fractionation and antibody uptake experiments showed that Cripto guides the Nodal precursor in detergent-resistant membranes to endocytic microdomains marked by GFP-Flotillin. We conclude that Nodal processing and endocytosis are coupled in signal-receiving cells.

The 20S proteasome is the catalytic core of the 26S proteasome. It comprises four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). Recent studies indicated that proteasome-specific chaperones and beta-subunit appendages assist in the formation of alpha-rings and dimerization of half-proteasomes, but the process involved in the assembly of beta-rings is poorly understood. Here, we clarify the mechanism of beta-ring formation on alpha-rings by characterizing assembly intermediates accumulated in cells depleted of each beta-subunit. Starting from beta2, incorporation of beta-subunits occurs in an orderly manner dependent on the propeptides of beta2 and beta5, and the C-terminal tail of beta2. Unexpectedly, hUmp1, a chaperone functioning at the final assembly step, is incorporated as early as beta2 and is required for the structural integrity of early assembly intermediates. We propose a model in which beta-ring formation is assisted by both intramolecular and extrinsic chaperones, whose roles are partially different between yeast and mammals.

Bacterial autotransporters are comprised of an N-terminal 'passenger domain' and a C-terminal beta barrel ('beta domain') that facilitates transport of the passenger domain across the outer membrane. Following translocation, the passenger domains of some autotransporters are cleaved by an unknown mechanism. Here we show that the passenger domain of the Escherichia coli O157:H7 autotransporter EspP is released in a novel autoproteolytic reaction. After purification, the uncleaved EspP precursor underwent proteolytic processing in vitro. An analysis of protein topology together with mutational studies strongly suggested that the reaction occurs inside the beta barrel and revealed that two conserved residues, an aspartate within the beta domain (Asp(1120)) and an asparagine (Asn(1023)) at the P1 position of the cleavage junction, are essential for passenger domain cleavage. Interestingly, these residues were also essential for the proteolytic processing of two distantly related autotransporters. The data strongly suggest that Asp(1120) and Asn(1023) form an unusual catalytic dyad that mediates self-cleavage through the cyclization of the asparagine. Remarkably, a very similar mechanism has been proposed for the maturation of eukaryotic viral capsids.

C-tail-anchored (C-TA) proteins are anchored to specific organelle membranes by a single transmembrane segment (TMS) at the C-terminus, extruding the N-terminal functional domains into the cytoplasm in which the TMS and following basic segment function as the membrane-targeting signals. Here, we analyzed the import route of mitochondrial outer membrane (MOM) C-TA proteins, Bak, Bcl-XL, and Omp25, using digitonin-permeabilized HeLa cells, which provide specific and efficient import under competitive conditions. These experiments revealed that (i) C-TA proteins were imported to the MOM through a common pathway independent of the components of the preprotein translocase of the outer membrane, (ii) the C-TA protein-targeting signal functioned autonomously in the absence of cytoplasmic factors that specifically recognize the targeting signals and deliver the preproteins to the MOM, (iii) the function of a cytoplasmic chaperone was required if the cytoplasmic domains of the C-TA proteins assumed an import-incompetent conformation, and intriguingly, (iv) the MOM-targeting signal of Bak, in the context of the Bak molecule, required activation by the interaction of its cytoplasmic domain with VDAC2 before MOM targeting.

We report the antiapoptotic effect of RNA-binding protein HuR, a critical regulator of the post-transcriptional fate of target transcripts. Among the most prominent mRNAs complexing with HuR is that encoding prothymosin alpha (ProTalpha), an inhibitor of the apoptosome. In HeLa cells, treatment with the apoptotic stimulus ultraviolet light (UVC) triggered the mobilization of ProTalpha mRNA to the cytoplasm and onto heavier polysomes, where its association with HuR increased dramatically. Analysis of a chimeric ProTalpha mRNA directly implicated HuR in regulating ProTalpha production: ProTalpha translation and cytoplasmic concentration increased in HuR-overexpressing cells and declined in cells in which HuR levels were lowered by RNA interference. Importantly, the antiapoptotic influence engendered by HuR was vitally dependent on ProTalpha expression, since use of oligomers that blocked ProTalpha translation abrogated the protective effect of HuR. Together, our data support a regulatory scheme whereby HuR binds the ProTalpha mRNA, elevates its cytoplasmic abundance and translation, and thereby elicits an antiapoptotic program.

The Toc core complex consists of the pore-forming Toc75 and the GTPases Toc159 and Toc34. We confirm that the receptor form of Toc159 is integrated into the membrane. The association of Toc34 to Toc75/Toc159 is GTP dependent and enhanced by preprotein interaction. The N-terminal half of the pSSU transit peptide interacts with high affinity with Toc159, whereas the C-terminal part stimulates its GTP hydrolysis. The phosphorylated C-terminal peptide of pSSU interacts strongly with Toc34 and therefore inhibits binding and translocation of pSSU into Toc proteoliposomes. In contrast, Toc159 recognises only the dephosphorylated forms. The N-terminal part of the pSSU presequence does not influence binding to the Toc complex, but is able to block import into proteoliposomes through its interaction with Toc159. We developed a model of differential presequence recognition by Toc34 and Toc159.

Preproteins with N-terminal presequences are imported into mitochondria at translocation contact sites that include the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). Little is known about the functional cooperation of these translocases. We have characterized translocation contact sites by a productive TOM-TIM-preprotein supercomplex to address the role of three translocase subunits that expose domains to the intermembrane space (IMS). The IMS domain of the receptor Tom22 is required for stabilization of the translocation contact site supercomplex. Surprisingly, the N-terminal segment of the channel Tim23, which tethers the TIM23 complex to the outer membrane, is dispensable for both protein import and generation of the TOM-TIM supercomplex. Tim50, with its large IMS domain, is crucial for generation but not for stabilization of the supercomplex. Thus, Tim50 functions as a dynamic factor and the IMS domain of Tom22 represents a stabilizing element in formation of a productive translocation contact site supercomplex.

Proteins of the intermembrane space (IMS) of mitochondria are typically synthesized without presequences. Little is known about their topogenesis. We used Tim13, a member of the 'small Tim protein' family, as model protein to investigate the mechanism of translocation into the IMS. Tim13 contains four conserved cysteine residues that bind a zinc ion as cofactor. Import of Tim13 did not depend on the membrane potential or ATP hydrolysis. Upon import into mitochondria Tim13 adopted a stably folded conformation in the IMS. Mutagenesis of the cysteine residues or pretreatment with metal chelators interfered with folding of Tim13 in vitro and impaired its import into mitochondria. Upon depletion of metal ions or modification of cysteine residues, imported Tim13 diffused back out of the IMS. We propose an import pathway in which (1) Tim13 can pass through the TOM complex into and out of the IMS in an unfolded conformation, and (2) cofactor acquisition stabilizes folding on the trans side of the outer membrane and traps Tim13 in the IMS, and drives unidirectional movement of the protein across the outer membrane of mitochondria.

We have recently identified a novel basal transcription complex, TAC, that is present and active in embryonal carcinoma (EC) cells but not in other adult cells such as COS7. In the search for factors involved in TAC formation, we found that expression of the adenoviral 12S E1A oncoprotein abolishes TAC formation in EC cells. This effect of E1A depends on its N-terminal domain that is essential for cell differentiation and that targets the transcriptional coactivators p300 and PCAF. Expression of p300 lacking its major E1A interaction domain, CH3, restores TAC formation in the presence of E1A, in a bromodomain- and HAT domain-dependent manner. Consistently, the unprocessed TFIIAalphabeta precursor that is selectively assembled into TAC is acetylated preferentially compared with the processed subunits present in 'free' TFIIA. Intriguingly, expression of p300 in COS7 cells that do not contain detectable levels of TAC instigates formation of TAC from endogenous components. Our data suggest that p300 plays a role in formation of the TBP-TFIIA-containing basal transcription complex, TAC.

The endoplasmic (ER) quality control apparatus ensures that misfolded or unassembled proteins are not deployed within the cell, but are retained in the ER and degraded. A glycoprotein-specific system involving the ER lectins calnexin and calreticulin is well documented, but very little is known about mechanisms that may operate for non-glycosylated proteins. We have used a folding mutant of a non- glycosylated membrane protein, proteolipid protein (PLP), to examine the quality control of this class of polypeptide. We find that calnexin associates with newly synthesized PLP molecules, binding stably to misfolded PLP. Calnexin also binds stably to an isolated transmembrane domain of PLP, suggesting that this chaperone is able to monitor the folding and assembly of domains within the ER membrane. Notably, this glycan-independent interaction with calnexin significantly retards the degradation of misfolded PLP. We propose that calnexin contributes to the quality control of non-glycosylated polytopic membrane proteins by binding to misfolded or unassembled transmembrane domains, and discuss our findings in relation to the role of calnexin in the degradation of misfolded proteins.

Communication between G protein-coupled receptor (GPCR) and epidermal growth factor receptor (EGFR) signalling systems involves cell surface proteolysis of EGF-like precursors. The underlying mechanisms of EGFR signal transactivation pathways, however, are largely unknown. We demonstrate that in squamous cell carcinoma cells, stimulation with the GPCR agonists LPA or carbachol specifically results in metalloprotease cleavage and release of amphiregulin (AR). Moreover, AR gene silencing by siRNA or inhibition of AR biological activity by neutralizing antibodies and heparin prevents GPCR-induced EGFR tyrosine phosphorylation, downstream mitogenic signalling events, cell proliferation, migration and activation of the survival mediator Akt/PKB. Therefore, despite some functional redundancy among EGF family ligands, the present study reveals a distinct and essential role for AR in GPCR-triggered cellular responses. Furthermore, we present evidence that blockade of the metalloprotease-disintegrin tumour necrosis factor-alpha-converting enzyme (TACE) by the tissue inhibitor of metalloprotease-3, a dominant-negative TACE mutant or RNA interference suppresses GPCR-stimulated AR release, EGFR activation and downstream events. Thus, TACE can function as an effector of GPCR-mediated signalling and represents a key element of the cellular receptor cross-talk network.

The ATPase SecA mediates post-translational translocation of precursor proteins through the SecYEG channel of the bacterial inner membrane. We show that SecA, up to now considered to be a stable dimer, is actually in equilibrium with a small fraction of monomers. In the presence of membranes containing acidic phospholipids or in certain detergents, SecA completely dissociates into monomers. A synthetic signal peptide also affects dissociation into monomers. In addition, conversion into the monomeric state can be achieved by mutating a small number of residues in a dimeric and fully functional SecA fragment. This monomeric SecA fragment still maintains strong binding to SecYEG in the membrane as well as significant in vitro translocation activity. Together, the data suggest that the SecA dimer dissociates during protein translocation. Since SecA contains all characteristic motifs of a certain class of monomeric helicases, and since mutations in residues shared with the helicases abolish its translocation activity, SecA may function in a similar manner.

The gene cyaB1 from the cyanobacterium Anabaena sp. PCC 7120 codes for a protein consisting of two N-terminal GAF domains (GAF-A and GAF-B), a PAS domain and a class III adenylyl cyclase catalytic domain. The catalytic domain is active as a homodimer, as demonstrated by reconstitution from complementary inactive point mutants. The specific activity of the holoenyzme increased exponentially with time because the product cAMP activated dose dependently and nucleotide specifically (half-maximally at 1 microM), identifying cAMP as a novel GAF domain ligand. Using point mutants of either the GAF-A or GAF-B domain revealed that cAMP activated via the GAF-B domain. We replaced the cyanobacterial GAF domain ensemble in cyaB1 with the tandem GAF-A/GAF-B assemblage from the rat cGMP-stimulated phosphodiesterase type 2, and converted cyaB1 to a cGMP-stimulated adenylyl cyclase. This demonstrated the functional conservation of the GAF domain ensemble since the divergence of bacterial and eukaryotic lineages >2 billion years ago. In cyanobacteria, cyaB1 may act as a cAMP switch to stabilize committed developmental decisions.

The endoplasmic reticulum (ER) is recognized as an important site for regulating cell surface expression of membrane proteins. We recently reported that only a fraction of newly synthesized delta opioid receptors could leave the ER and reach the cell surface, the rest being degraded by proteasomes. Here, we demonstrate that membrane-permeable opioid ligands facilitate maturation and ER export of the receptor, thus acting as pharmacological chaperones. We propose that these ligands stabilize the newly synthesized receptor in the native or intermediate state of its folding pathway, possibly by inducing stabilizing conformational constrains within the hydrophobic core of the protein. The receptor precursors that are retained in the ER thus represent fully competent folding intermediates that can be targets for pharmacological intervention aimed at regulating receptor expression and cellular responsiveness. The pharmacological chaperone action is independent of the intrinsic signaling efficacy of the ligand, since both agonists and antagonists were found to promote receptor maturation. This novel property of G protein-coupled receptor ligands may have important implications when considering their effects on cellular responsiveness during therapeutic treatments.

We raised monoclonal antibodies against senile plaque (SP) amyloid and obtained a clone 9D2, which labeled amyloid fibrils in SPs and reacted with approximately 50/100 kDa polypeptides in Alzheimer's disease (AD) brains. We purified the 9D2 antigens and cloned a cDNA encoding its precursor, which was a novel type II transmembrane protein specifically expressed in neurons. This precursor harbored three collagen-like Gly-X-Y repeat motifs and was partially homologous to collagen type XIII. Thus, we named the 9D2 antigen as CLAC (collagen-like Alzheimer amyloid plaque component), and its precursor as CLAC-P/collagen type XXV. The extracellular domain of CLAC-P/collagen type XXV was secreted by furin convertase, and the N-terminus of CLAC deposited in AD brains was pyroglutamate modified. Both secreted and membrane-tethered forms of CLAC-P/collagen type XXV specifically bound to fibrillized Abeta, implicating these proteins in beta-amyloidogenesis and neuronal degeneration in AD.

Escherichia coli preprotein translocase comprises a membrane-embedded trimeric complex of SecY, SecE and SecG. Previous studies have shown that this complex forms ring-like assemblies, which are thought to represent the preprotein translocation channel across the membrane. We have analyzed the functional state and the quaternary structure of the SecYEG translocase by employing cross-linking and blue native gel electrophoresis. The results show that the SecYEG monomer is a highly dynamic structure, spontaneously and reversibly associating into dimers. SecG-dependent tetramers and higher order SecYEG multimers can also exist in the membrane, but these structures form at high SecYEG concentration or upon overproduction of the complex only. The translocation process does not affect the oligomeric state of the translocase and arrested preproteins can be trapped with SecYEG or SecYE dimers. Dissociation of the dimer into a monomer by detergent induces release of the trapped preprotein. These results provide direct evidence that preproteins cross the bacterial membrane, associated with a translocation channel formed by a dimer of SecYEG.

The human transcription factor Oct-1 can stimulate transcription from a variety of promoters by interacting with the coactivators OBF-1/OCA-B/BOB-1, SNAP190 and VP16. These proteins contact Oct-1 regions different from the DNA binding surface. Oct-1 also stimulates the DNA replication of adenovirus through its DNA binding site in the origin. The Oct-1 POU homeodomain (POUhd) binds the adenovirus precursor terminal protein pTP, which serves as the protein primer of DNA replication and recruits pTP to the origin. To map the interaction with pTP at the POUhd surface, we screened a library of randomly mutated POU domains and identified mutations that interfered with pTP interaction and DNA replication stimulation. These mutants clustered at a surface different from those recognized by OBF-1, SNAP190 and VP16. Unexpectedly, the pTP binding region largely overlapped with the DNA binding surface of POUhd. In agreement with this, pTP binding and DNA binding were mutually exclusive. We propose a model to reconcile pTP recruitment and DNA binding by Oct-1.

Cell-binding and internalization studies on neuronal and non-neuronal cells have demonstrated that the 37-kDa/67-kDa laminin receptor (LRP/LR) acts as the receptor for the cellular prion protein (PrP). Here we identify direct and heparan sulfate proteoglycan (HSPG)-dependent interaction sites mediating the binding of the cellular PrP to its receptor, which we demonstrated in vitro on recombinant proteins. Mapping analyses in the yeast two-hybrid system and cell-binding assays identified PrPLRPbd1 [amino acids (aa) 144-179] as a direct and PrPLRPbd2 (aa 53-93) as an indirect HSPG-dependent laminin receptor precursor (LRP)-binding site on PrP. The yeast two-hybrid system localized the direct PrP-binding domain on LRP between aa 161 and 179. Expression of an LRP mutant lacking the direct PrP-binding domain in wild-type and mutant HSPG-deficient Chinese hamster ovary cells by the Semliki Forest virus system demonstrates a second HSPG-dependent PrP-binding site on LRP. Considering the absence of LRP homodimerization and the direct and indirect LRP-PrP interaction sites, we propose a comprehensive model for the LRP-PrP-HSPG complex.

Recently, we identified the 37-kDa laminin receptor precursor (LRP) as an interactor for the prion protein (PrP). Here, we show the presence of the 37-kDa LRP and its mature 67-kDa form termed high-affinity laminin receptor (LR) in plasma membrane fractions of N2a cells, whereas only the 37-kDa LRP was detected in baby hamster kidney (BHK) cells. PrP co-localizes with LRP/LR on the surface of N2a cells and Semliki Forest virus (SFV) RNA transfected BHK cells. Cell-binding assays reveal the LRP/LR-dependent binding of cellular PrP by neuronal and non-neuronal cells. Hyperexpression of LRP on the surface of BHK cells results in the binding of exogenous PrP. Cell binding is similar in PrP(+/+) and PrP(0/0) primary neurons, demonstrating that PrP does not act as a co-receptor of LRP/LR. LRP/LR-dependent internalization of PrP is blocked at 4 degrees C. Secretion of an LRP mutant lacking the transmembrane domain (aa 86-101) from BHK cells abolishes PrP binding and internalization. Our results show that LRP/LR acts as the receptor for cellular PrP on the surface of mammalian cells.

Proteins of Gram-negative bacteria destined to the extracellular milieu must cross the two cellular membranes and then fold at the appropriate time and place. The synthesis of a precursor may be a strategy to maintain secretion competence while preventing aggregation or premature folding (especially for large proteins). The secretion of 230 kDa filamentous haemagglutinin (FHA) of Bordetella pertussis requires the synthesis and the maturation of a 367 kDa precursor that undergoes the proteolytic removal of its approximately 130 kDa C-terminal intramolecular chaperone domain. We have identified a specific protease, SphB1, responsible for the timely maturation of the precursor FhaB, which allows for extracellular release of FHA. SphB1 is a large exported protein with a subtilisin-like domain and a C-terminal domain typical of bacterial autotransporters. SphB1 is the first described subtilisin-like protein that serves as a specialized maturation protease in a secretion pathway of Gram-negative bacteria. This is reminiscent of pro-protein convertases of eukaryotic cells.

Influenza haemagglutinin (HA) is responsible for fusing viral and endosomal membranes during virus entry. In this process, conformational changes in the HA relocate the HA(2) N-terminal 'fusion peptide' to interact with the target membrane. The highly conserved HA fusion peptide shares composition and sequence features with functionally analogous regions of other viral fusion proteins, including the presence and distribution of glycines and large side-chain hydrophobic residues. HAs with mutations in the fusion peptide were expressed using vaccinia virus recombinants to examine the requirement for fusion of specific hydrophobic residues and the significance of glycine spacing. Mutant HAs were also incorporated into infectious influenza viruses for analysis of their effects on infectivity and replication. In most cases alanine, but not glycine substitutions for the large hydrophobic residues, yielded fusion-competent HAs and infectious viruses, suggesting that the conserved spacing of glycines may be structurally significant. When viruses containing alanine substitutions for large hydrophobic residues were passaged, pseudoreversion to valine was observed, indicating a preference for large hydrophobic residues at specific positions. Viruses were also obtained with serine, leucine or phenylalanine as the N-terminal residue, but these replicated to significantly lower levels than wild-type virus with glycine at this position.

Hepadnaviruses do not infect cultured cells, therefore our knowledge of the mechanism of the early stages of virus-cell interaction is rather poor. In this study, we show that dimethylsulfoxide (DMSO)-treated HepG2 hepatoblastoma cells are infected efficiently by serum-derived hepatitis B virus (HBV) as monitored by viral gene expression and replication markers. To measure virus attachment, a variety of HBV surface proteins (HBsAgs) were conjugated to polystyrene beads and their capacity to attach cells was visualized and quantified by light microscopy at a single-cell resolution. Remarkably, DMSO increases the attachment efficiency by >200-fold. We further identify the QLDPAF sequence within preS1 as the receptor-binding viral domain epitope. Interestingly, a similar sequence is shared by several cellular, bacterial and viral proteins involved in cell adhesion, attachment and fusion. We also found that the small HBsAg contains a secondary attachment site that recognizes a distinct receptor on the cell membrane. Furthermore, we provide evidence in support of multivalent HBV attachment with synergistic interplay. Our data depict a mechanistic view of virus attachment and ingestion.

Islet cell autoantigen (ICA) 512 is a receptor-tyrosine phosphatase-like protein associated with the secretory granules of neuroendocrine cells, including pancreatic beta-cells. Binding of its cytoplasmic tail to beta2-syntrophin suggests that ICA512 connects secretory granules to the utrophin complex and the actin cytoskeleton. Here we show that stimulation of insulin secretion from INS-1 cells triggers the biosynthesis of pro-ICA512 and the degradation of its mature form. Inhibition of calpain, which is activated upon stimulation of insulin secretion, prevents the Ca2+-dependent proteolysis of ICA512. In vitro mu-calpain cleaves ICA512 between a putative PEST domain and the beta2-syntrophin binding site, whereas binding of ICA512 to beta2-syntrophin protects the former from cleavage. beta2-syntrophin and its F-actin-binding protein utrophin are enriched in subcellular fractions containing secretory granules. ICA512 preferentially binds phospho-beta2-syntrophin and stimulation of insulin secretion induces the Ca2+-dependent, okadaic acid-sensitive dephosphorylation of beta2-syntrophin. Similarly to calpeptin, okadaic acid inhibits ICA512 proteolysis and insulin secretion. Thus, stimulation of insulin secretion might promote the mobilization of secretory granules by inducing the dissociation of ICA512 from beta2-syntrophin-utrophin complexes and the cleavage of the ICA512 cytoplasmic tail by mu-calpain.

Unfolding is an essential process during translocation of preproteins into mitochondria; however, controversy exists as to whether mitochondria play an active role in unfolding. We have established an in vitro system with a kinetic saturation of the mitochondrial import machinery, yielding translocation rates comparable to in vivo import rates. Preproteins with short N-terminal segments in front of a folded domain show a characteristic delay of the onset of translocation (lag phase) although the maximal import rate is similar to that of longer preproteins. The lag phase is shortened by extending the N-terminal segment to improve the accessibility to matrix heat shock protein 70 and abolished by unfolding of the preprotein. A mutant mtHsp70 defective in binding to the inner membrane prolongs the lag phase and reduces the translocation activity. A direct comparison of the rate of spontaneous unfolding in solution with that during translocation demonstrates that unfolding by mitochondria is significantly faster, proving an active unfolding process. We conclude that access of mtHsp70 to N-terminal preprotein segments is critical for active unfolding and initiation of translocation.

The ADP/ATP carrier (AAC) is a major representative of mitochondrial preproteins lacking an N-terminal presequence. AAC contains targeting information in each of its three modules, which has led to a search for the dominant targeting region. An alternative, not yet tested model would be that several distinct targeting signals function simultaneously in import of the preprotein. We report that the three AAC modules cooperate in binding to the receptor Tom70 such that three Tom70 dimers are recruited to one preprotein. The modules are transferred to the import pore in a stepwise manner and cooperate again in the accumulation of AAC in the general import pore complex. AAC can cross the outer membrane with an internal segment first, i.e. in a loop formation. Each module of AAC is required for dimerization in the inner membrane. We propose a new concept for import of the hydrophobic carrier proteins into mitochondria where multiple signals cooperate in receptor recruitment, outer membrane translocation via loop formation and assembly in the inner membrane.

A multisubunit complex in the mitochondrial outer membrane, the TOM complex, mediates targeting and membrane translocation of nuclear-encoded preproteins. We have isolated the TOM holo complex, containing the preprotein receptor components Tom70 and Tom20, and the TOM core complex, which lacks these receptors. The interaction of recombinant mitochondrial preproteins with both types of soluble TOM complex was analyzed. Preproteins bound efficiently in a specific manner to the isolated complexes in the absence of chaperones and lipids in a bilayer structure. Using fluorescence correlation spectroscopy, a dissociation constant in the nanomolar range was determined. The affinity was lower when the preprotein was stabilized in its folded conformation. Following the initial binding, the presequence was transferred into the translocation pore in a step that required unfolding of the mature part of the preprotein. This translocation step was also mediated by protease-treated TOM holo complex, which contains almost exclusively Tom40. Thus, the TOM core complex, consisting of Tom40, Tom22, Tom6 and Tom7, is a molecular machine that can recognize and partially translocate mitochondrial precursor proteins.

Spindle pole bodies (SPBs) are the centrosome equivalents in yeast, required for microtubule organization. In yeast, the SPB further serves as the attachment sites of the prospore membrane during meiosis. Here we report the identification of two new meiosis-specific components of the SPB, Mpc54p and Mpc70p, and the first protein specific for the prospore membrane, Don1p. Mpc54p and Mpc70p are not present in mitotic SPBs, and during meiosis II they are components of a meiosis-specific structural alteration of the outer plaque of the SPB. Both proteins are dispensable for the meiotic divisions but are essentially required for the formation of the prospore membrane. In the mpc54 and mpc70 mutants, the Don1p-containing precursors of the prospore membrane can still be found in the cytoplasm and associated with the SPB. Unexpectedly, however, the assembly of the precursors to a continuous membrane system is affected. Thus, the meiotic SPB is directly involved in the formation of a specialized membrane system, the membrane of the prospore.

Processing of the p105 precursor to form the active subunit p50 of the NF-kappaB transcription factor is a unique case in which the ubiquitin system is involved in limited processing rather than in complete destruction of the target substrate. A glycine-rich region along with a downstream acidic domain have been demonstrated to be essential for processing. Here we demonstrate that following IkappaB kinase (IkappaK)-mediated phosphorylation, the C-terminal domain of p105 (residues 918-934) serves as a recognition motif for the SCF(beta)(-TrCP) ubiquitin ligase. Expression of IkappaKbeta dramatically increases processing of wild-type p105, but not of p105-Delta918-934. Dominant-negative beta-TrCP inhibits IkappaK-dependent processing. Furthermore, the ligase and wild-type p105 but not p105-Delta918-934 associate physically following phosphorylation. In vitro, SCF(beta)(-TrCP) specifically conjugates and promotes processing of phosphorylated p105. Importantly, the TrCP recognition motif in p105 is different from that described for IkappaBs, beta-catenin and human immunodeficiency virus type 1 Vpu. Since p105-Delta918-934 is also conjugated and processed, it appears that p105 can be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs.

Aminopeptidase I (API) is imported into the yeast vacuole/lysosome by a constitutive non-classical vesicular transport mechanism, the cytoplasm to vacuole targeting (Cvt) pathway. Newly synthesized precursor API is sequestered in double-membrane cytoplasmic Cvt vesicles. The Cvt vesicles fuse with the vacuole, releasing single-membrane Cvt bodies containing proAPI into the vacuolar lumen, and maturation of API occurs when the Cvt body is degraded, releasing mature API. Under starvation conditions, API is transported to the vacuole by macroautophagy, an inducible, non-selective mechanism that shares many similarities with the Cvt pathway. Here we show that Tlg2p, a member of the syntaxin family of t-SNARE proteins, and Vps45p, a Sec1p homologue, are required in the constitutive Cvt pathway, but not in inducible macroautophagy. Fractionation and protease protection experiments indicate that Tlg2p is required prior to or at the step of API segregation into the Cvt vesicle. Thus, the early Vps45-Tlg2p-dependent step of the Cvt pathway appears to be mechanistically distinct from the comparable stage in macroautophagy. Vps45p associates with both the Tlg2p and Pep12p t-SNAREs, but API maturation is not blocked in a pep12(ts) mutant, indicating that Vps45p independently regulates the function of multiple t-SNARES at distinct trafficking steps.

The NF-kappaB precursor p105 has dual functions: cytoplasmic retention of attached NF-kappaB proteins and generation of p50 by processing. It is poorly understood whether these activities of p105 are responsive to signalling processes that are known to activate NF-kappaB p50-p65. We propose a model that p105 is inducibly degraded, and that its degradation liberates sequestered NF-kappaB subunits, including its processing product p50. p50 homodimers are specifically bound by the transcription activator Bcl-3. We show that TNFalpha, IL-1beta or phorbolester (PMA) trigger rapid formation of Bcl-3-p50 complexes with the same kinetics as activation of p50-p65 complexes. TNF-alpha-induced Bcl-3-p50 formation requires proteasome activity, but is independent of p50-p65 released from IkappaBalpha, indicating a pathway that involves p105 proteolysis. The IkappaB kinases IKKalpha and IKKbeta physically interact with p105 and inducibly phosphorylate three C-terminal serines. p105 is degraded upon TNF-alpha stimulation, but only when the IKK phospho-acceptor sites are intact. Furthermore, a p105 mutant, lacking the IKK phosphorylation sites, acts as a super-repressor of IKK-induced NF-kappaB transcriptional activity. Thus, the known NF-kappaB stimuli not only cause nuclear accumulation of p50-p65 heterodimers but also of Bcl-3-p50 and perhaps further transcription activator complexes which are formed upon IKK-mediated p105 degradation.

The major coat proteins of clathrin-coated vesicles are the clathrin triskelion and heterotetrameric associated protein (AP) complexes. The APs are thought to be involved in cargo capture and recruitment of clathrin to the membrane during endocytosis and sorting in the trans-Golgi network/endosomal system. AP180 is an abundant coat protein in brain clathrin-coated vesicles, and it has potent clathrin assembly activity. In Saccharomyces cerevisiae, there are 13 genes encoding homologs of heterotetrameric AP subunits and two genes encoding AP180-related proteins. To test the model that clathrin function is dependent on the heterotetrameric APs and/or AP180 homologs, yeast strains containing multiple disruptions in AP subunit genes, as well as in the two YAP180 genes, were constructed. Surprisingly, the AP deletion strains did not display the phenotypes associated with clathrin deficiency, including slowed growth and endocytosis, defective late Golgi protein retention and impaired cytosol to vacuole/autophagy function. Clathrin-coated vesicles isolated from multiple AP deletion mutants were morphologically indistinguishable from those from wild-type cells. These results indicate that clathrin function and recruitment onto membranes are not dependent upon heterotetrameric adaptors or AP180 homologs in yeast. Therefore, alternative mechanisms for clathrin assembly and coated vesicle formation, as well as the role of AP complexes and AP180-related proteins in these processes, must be considered.

We have analysed the structural organization of the TIM17.23 complex, the preprotein translocase of the mitochondrial inner membrane specific for protein targeting to the matrix. The components Tim17, Tim23 and Tim44 are present in this complex in equimolar amounts. A sub-complex containing Tim23 and Tim44 but no Tim17, or a sub-complex containing Tim23 and Tim17 but no Tim44 was not detected. Tim44 is peripherally associated at the matrix side. Tim44 forms dimers which recruit two molecules of mt-Hsp70 to the sites of protein import. A sequential, hand-over-hand mode of interaction of these two mt-Hsp70.Tim44 complexes with a translocating polypeptide chain is proposed.

Glycosylphosphatidylinositols (GPI) are essential components in the plasma membrane of the protozoan parasite Leishmania mexicana, both as membrane anchors for the major surface macromolecules and as the sole class of free glycolipids. We provide evidence that L.mexicana dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis, is localized to a distinct tubular subdomain of the endoplasmic reticulum (ER), based on the localization of a green fluorescent protein (GFP)-DPMS chimera and subcellular fractionation experiments. This tubular membrane (termed the DPMS tubule) is also enriched in other enzymes involved in GPI biosynthesis, can be specifically stained with the fluorescent lipid, BODIPY-C5-ceramide, and appears to be connected to specific subpellicular microtubules that underlie the plasma membrane. Perturbation of microtubules and DPMS tubule structure in vivo results in the selective accumulation of GPI anchor precursors, but not free GPIs. The DPMS tubule is closely associated morphologically with the single Golgi apparatus in non-dividing and dividing cells, appears to exclude luminal ER resident proteins and is labeled, together with the Golgi apparatus, with another GFP chimera containing the heterologous human Golgi marker beta1,2-N-acetylglucosaminyltransferase-I. The possibility that the DPMS-tubule is a stable transitional ER is discussed.

prlA mutations in the gene encoding the SecY subunit of the membrane domain of the Escherichia coli preprotein translocase confer many phenotypes: enhanced translocation rates, increased affinity for SecA, diminished requirement for functional leader sequences, reduced proton-motive force (PMF) dependence of preprotein translocation and facilitated translocation of preproteins with folded domains. We now report that both prlA and prlG mutations weaken the associations between the SecY, SecE and SecG subunits of the translocase. This loosened association increases the initiation of translocation by facilitating the insertion of SecA with its bound preprotein but reduces the stimulatory effect of the PMF during the initial step of translocation. Furthermore, the originally isolated prlA4 mutant, which possesses a particularly labile SecYEG complex, acquired a secondary mutation that restored the stability while conserving the flexibility of the complex. Combinations of certain prlA and prlG mutations, known to cause synthetic lethality in vivo, dramatically loosen subunit association and lead to complete disassembly of SecYEG. These findings underscore the importance of the loosened SecYEG association for the Prl phenotypes. We propose a model in which each of the PrlA and PrlG phenotypes derive from this enhanced SecYEG conformational flexibility.

Members of the mitochondrial carrier family such as the ADP/ATP carrier (AAC) are composed of three structurally related modules. Here we show that each of the modules contains a mitochondrial import signal recognized by Tim10 and Tim12 in the intermembrane space. The first and the second module are translocated across the outer membrane independently of the membrane potential, DeltaDeltapsipsi, but they are not inserted into the inner membrane. The third module interacts tightly with the TOM complex and thereby prevents complete translocation of the precursor across the outer membrane. At this stage, binding of a TIM9.10 complex confers a topology to the translocation intermediate which reflects the modular structure of the AAC. The precursor is then transferred to the TIM9.10.12 complex, still interacting with the TOM complex. Release of the precursor from the TOM complex and insertion into the inner membrane by the TIM22.54 complex requires a DeltaDeltapsipsi-responsive signal in the third module.

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation.

The ectodomains of many proteins located at the cell surface are shed upon cell stimulation. One such protein is the heparin-binding EGF-like growth factor (HB-EGF) that exists in a membrane-anchored form which is converted to a soluble form upon cell stimulation with TPA, an activator of protein kinase C (PKC). We show that PKCdelta binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin-gamma/ADAM9, a member of the metalloprotease-disintegrin family. Furthermore, the presence of constitutively active PKCdelta or MDC9 results in the shedding of the ectodomain of proHB-EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase-negative PKCdelta, suppress the TPA-induced shedding of the ectodomain. These results suggest that MDC9 and PKCdelta are involved in the stimulus-coupled shedding of the proHB-EGF ectodomain.

Nuclear-encoded mitochondrial matrix proteins in most cases contain N-terminal targeting signals and are imported in a linear N- to C-terminal (N-->C) fashion. We asked whether import can also occur in a C- to N-terminal direction (C-->N). We placed targeting signals at the C-terminus of passenger proteins. Import did occur in this 'backwards' fashion. It paralleled that of the 'normal' N-->C mechanism in terms of efficiency, rate, energetic requirements and ability to mediate unfolding and refolding during and following import of protein containing a folded domain. Furthermore, this reaction was mediated by the TIM17-23 machinery. The import pathway taken by certain inner-membrane proteins contains elements of such a C-->N translocation pathway, as they are targeted to mitochondria by internal targeting signals. These internal targeting signals appear to form loop structures together with neighbouring transmembrane segments, and penetrate the inner membrane in a membrane-potential-dependent manner. The dimeric TIM17-23 complex, together with mt-Hsp70, acts on both sides of the loop structure to facilitate their translocation into the matrix. On one side of the loop import occurs in the common N-->C direction, whereas the translocation of the other side involves the novel C-->N import direction. We conclude therefore that the mitochondrial import machinery displays no preference for the directionality of the import process.

Unfolding of preproteins and translocation across the mitochondrial membranes requires their interaction with mt-Hsp70 and Tim44 at the inner face of the inner membrane and ATP as an energy source. We measured the temperature dependence of the rates of unfolding and import into the matrix of two folded passenger domains, the tightly folded heme-binding domain (HBD) of cytochrome b2 and the loosely folded mouse dihydrofolate reductase (DHFR). Despite the stability of the HBD, its rates of thermal breathing were fast and the preprotein was imported rapidly at all temperatures. In contrast, rates of unfolding and import of DHFR were strongly temperature dependent and import was significantly slower than unfolding. In addition, import rates of DHFR were strongly dependent on the length of the presequence. We propose that the mitochondrial import motor does not exert a constant pulling force. Rather, mt-Hsp70 appears to release a translocating polypeptide chain such that the precursor can then slide back and refold on the surface of the mitochondria. Refolding competes with translocation, and passengers may undergo several rounds of unfolding and refolding prior to their import.

NF-kappaB activation in response to UV irradiation of HeLa cells or of primary human skin fibroblasts occurs with two overlapping kinetics but totally different mechanisms. Although both mechanisms involve induced dissociation of NF-kappaB from IkappaBalpha and degradation of IkappaBalpha, targeting for degradation and signaling are different. Early IkappaBalpha degradation at 30 min to approximately 6 h is not initiated by UV-induced DNA damage. It does not require IkappaB kinase (IKK), as shown by introduction of a dominant-negative kinase subunit, and does not depend on the presence of the phosphorylatable substrate, IkappaBalpha, carrying serines at positions 32 and 36. Induced IkappaBalpha degradation requires, however, intact N- (positions 1-36) and C-terminal (positions 277-287) sequences. IkappaB degradation and NF-kappaB activation at late time points, 15-20 h after UV irradiation, is mediated through DNA damage-induced cleavage of IL-1alpha precursor, release of IL-1alpha and autocrine/paracrine action of IL-1alpha. Late-induced IkappaBalpha requires the presence of Ser32 and Ser36. The late mechanism indicates the existence of signal transfer from photoproducts in the nucleus to the cytoplasm. The release of the 'alarmone' IL-1alpha may account for some of the systemic effects of sunlight exposure.

Bone morphogenetic protein-4 (BMP-4) is a multifunctional developmental regulator. BMP-4 is synthesized as an inactive precursor that is proteolytically activated by cleavage following the amino acid motif -Arg-Ser-Lys-Arg-. Very little is known about processing and secretion of BMPs. The proprotein convertases (PCs) are a family of seven structurally related serine endoproteases, at least one of which, furin, cleaves after the amino acid motif -Arg-X-Arg/Lys-Arg-. To examine potential roles of PCs during embryonic development we have misexpressed a potent protein inhibitor of furin, alpha1-antitrypsin Portland (alpha1-PDX) in early Xenopus embryos. Ectopic expression of alpha1-PDX phenocopies the effect of blocking endogenous BMP activity, leading to dorsalization of mesoderm and direct neural induction. alpha1-PDX-mediated neural induction can be reversed by co-expression of downstream components of the BMP-4 signaling pathway. Thus, alpha1-PDX can block BMP activity upstream of receptor binding, suggesting that it inhibits an endogenous BMP-4 convertase(s). Consistent with this hypothesis, alpha1-PDX prevents cleavage of BMP-4 in an oocyte translation assay. Using an in vitro digestion assay, we demonstrate that four members of the PC family have the ability to cleave BMP-4, but of these, only furin and PC6B are sensitive to alpha1-PDX. These studies provide the first in vivo evidence that furin and/or PC6 proteolytically activate BMP-4 during vertebrate embryogenesis.

Sphingolipid activator proteins SAP-A, -B, -C and -D (also called saposins) are generated by proteolytic processing from a 73 kDa precursor and function as obligatory activators of lysosomal enzymes involved in glycosphingolipid metabolism. Although the SAP precursor can be recognized by the mannose-6-phosphate (M-6-P) receptor and shuttled directly from the secretory pathway to the lysosome, a substantial fraction of newly synthesized precursor is secreted from the cell where it may participate in sphingolipid transport and signaling events. Re-uptake of the secreted precursor is mediated by high-affinity cell surface receptors that are apparently distinct from the M-6-P receptor. We found that the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic receptor that is expressed on most cells, can mediate cellular uptake and lysosomal delivery of SAP precursor. Additional in vivo experiments in mice revealed that the mannose receptor system on macrophages also participates in precursor internalization. We conclude that SAP precursor gains entry into cells by at least three independent receptor mechanisms including the M-6-P receptor, the mannose receptor and LRP.

Mitochondrial precursor proteins with basic targeting signals may be transported across the outer membrane by sequential binding to acidic receptor sites of increasing affinity. To test this 'acid chain' hypothesis, we assayed the interaction of mitochondrial precursors with three acidic receptor domains: the cytosolic domain of Tom20 and the intermembrane space domain of Tom22 and Tim23. The apparent affinity and salt resistance of precursor binding increased in the order Tom20

We describe the identification of two Escherichia coli genes required for the export of cofactor-containing periplasmic proteins, synthesized with signal peptides containing a twin arginine motif. Both gene products are homologous to the maize HCF106 protein required for the translocation of a subset of lumenal proteins across the thylakoid membrane. Disruption of either gene affects the export of a range of such proteins, and a complete block is observed when both genes are inactivated. The Sec protein export pathway was unaffected, indicating the involvement of the gene products in a novel export system. The accumulation of active cofactor-containing proteins in the cytoplasm of the mutant strains suggests a role for the gene products in the translocation of folded proteins. One of the two HCF106 homologues is encoded by the first gene of a four cistron operon, tatABCD, and the second by an unlinked gene, tatE. A mutation previously assigned to the hcf106 homologue encoded at the tatABCD locus, mttA, lies instead in the tatB gene.