Presence Of Ribosomes Research Articles

Cytoplasmic ribosomes hitchhike on mitochondria to dendrites.

Neurons rely on local protein synthesis to rapidly modify the proteome of neurites distant from the cell body. A prerequisite for local protein synthesis is the presence of ribosomes in the neurite, but the mechanisms of ribosome transport in neurons remain poorly defined. Here, we find that ribosomes hitchhike on mitochondria for their delivery to the dendrite of a sensory neuron in C. elegans. Ribosomes co-transport with dendritic mitochondria, and their association requires the atypical Rho GTPase MIRO-1. Disrupting mitochondrial transport prevents ribosomes from reaching the dendrite, whereas ectopic re-localization of mitochondria results in a concomitant re-localization of ribosomes, demonstrating that mitochondria are required and sufficient for instructing ribosome distribution in dendrites. Endolysosomal organelles that are involved in mRNA transport and translation can associate with mitochondria and ribosomes but do not play a significant role in ribosome transport. These results reveal a mechanism for dendritic ribosome delivery, which is a critical upstream requirement for local protein synthesis.

Tau protein- induced sequestration of the eukaryotic ribosome: Implications in neurodegenerative disease

The human tau is a microtubule-associated intrinsically unstructured protein that forms intraneuronal cytotoxic deposits in neurodegenerative diseases, like tauopathies. Recent studies indicate that in Alzheimer’s disease, ribosomal dysfunction might be a crucial event in the disease pathology. Our earlier studies had demonstrated that amorphous protein aggregation in the presence of ribosome can lead to sequestration of the ribosomal components. The present study aims at determining the effect of incubation of the full-length tau protein (Ht40) and its microtubule binding 4-repeat domain (K18) on the eukaryotic ribosome. Our in vitro studies show that incubation of Ht40 and the K18 tau variants with isolated non-translating yeast ribosome can induce a loss of ribosome physical integrity resulting in formation of tau-rRNA-ribosomal protein aggregates. Incubation with the tau protein variants also led to a disappearance of the peak indicating the ribosome profile of the HeLa cell lysate and suppression of translation in the human in vitro translation system. The incubation of tau protein with the ribosomal RNA leads to the formation of tau-rRNA aggregates. The effect of K18 on the yeast ribosome can be mitigated in the presence of cellular polyanions like heparin and tRNA, thereby indicating the electrostatic nature of the aggregation process.

PpGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Gram-positive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Gram-positive bacterium Staphylococcus aureus We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases--RsgA, RbgA, Era, HflX, and ObgE--as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with an rsgA deletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs from Bacillus subtilis and Enterococcus faecalis retain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria.

A comparison of the folding characteristics of free and ribosome‐tethered polypeptide chains using limited proteolysis and mass spectrometry

The kinetics and thermodynamics of protein folding are commonly studied in vitro by denaturing/renaturing intact protein sequences. How these folding mechanisms relate to de novo folding that occurs as the nascent polypeptide emerges from the ribosome is much less well understood. Here, we have employed limited proteolysis followed by mass spectrometry analyses to compare directly free and ribosome-tethered polypeptide chains of the Src-homology 3 (SH3) domain and its unfolded variant, SH3-m10. The disordered variant was found to undergo faster proteolysis than SH3. Furthermore, the trypsin cleavage patterns observed show minor, but significant, differences for the free and ribosome-bound nascent chains, with significantly fewer tryptic peptides detected in the presence of ribosome. The results highlight the utility of limited proteolysis coupled with mass spectrometry for the structural analysis of these complex systems, and pave the way for detailed future analyses by combining this technique with chemical labeling methods (for example, hydrogen-deuterium exchange, photochemical oxidation) to analyze protein folding in real time, including in the presence of additional ribosome-associated factors.

182 Investigating the mode of action of an essential OBG GTPase, CgtA in bacteria

CgtA is an essential OBG GTPase (Trach & Hoch, 1989) highly conserved from bacteria to eukaryotes. It is a multifunctional protein, involved in DNA replication, chromosome partitioning (Slominska et al., 2002), nutritional stress response, initiation of sporulation, ribosome maturation, etc. Despite being a multifunctional essential protein, its mode of action is not well- characterized and key question remains: how does this protein work in wide varieties of cellular function? The expression of cgtA-mRNA increases on the onset of nutritional stress. Purified CgtA protein shows increased GTPase activity in the presence of ribosome. Our experiment with thiostrepton reveals that, although ribosome is able to trigger the GTPase activity of CgtA, its probable site of GTPase inducing activity is different from other regular translation factors like EF-G, that uses GTP. For structure function study we have generated an energy minimized homology model of the Vibrio cholerae CgtA protein, which reveals two large domains, an OBG-fold and a GTP– hydrolysis domain, with an extended C-terminal part. We compared the amino acid sequence of CgtA across various species in the database, and found that its Glycine98 and the Tyrosine195 residues are 100% conserved in prokaryotes. These amino acids are highly conserved in eukaryotes as well. Gly98 and Tyr195 are located on the hinge region of CgtA comprising of portions of the OBG and the GTP–hydrolysis domains, respectively. To decipher the mode of actions of CgtA and the role of the conserved Gly98 residue, we have replaced the Gly with a relatively larger amino acid, i.e. Asp. Our study reveals that the mutant CgtA(G98D) shows a reduced GTPase activity in presence of ribosome compared to the wild type. This indicates a restricted inter-domain movement of CgtA due to the above point mutation. To understand this phenomenon we are using MD simulations. We will discuss results from MD simulations and other mutation studies as well. Our results indicate that ribosome acts as a modulator for increasing the GTPase activity of CgtA. The perfect conservation of G98 residue is important for the proper functionality of CgtA.

Competitive Binding of the SecA ATPase and Ribosomes to the SecYEG Translocon

During co-translational membrane insertion of membrane proteins with large periplasmic domains, the bacterial SecYEG complex needs to interact both with the ribosome and the SecA ATPase. Although the binding sites for SecA and the ribosome overlap, it has been suggested that these ligands can interact simultaneously with SecYEG. We used surface plasmon resonance and fluorescence correlation spectroscopy to examine the interaction of SecA and ribosomes with the SecYEG complex present in membrane vesicles and the purified SecYEG complex present in a detergent-solubilized state or reconstituted into nanodiscs. Ribosome binding to the SecYEG complex is strongly stimulated when the ribosomes are charged with nascent chains of the monotopic membrane protein FtsQ. This binding is competed by an excess of SecA, indicating that binding of SecA and ribosomes to SecYEG is mutually exclusive.

Sordarin Derivatives Induce a Novel Conformation of the Yeast Ribosome Translocation Factor eEF2

The sordarins are fungal specific inhibitors of the translation factor eEF2, which catalyzes the translocation of tRNA and mRNA after peptide bond formation. We have determined the crystal structures of eEF2 in complex with two novel sordarin derivatives. In both structures, the three domains of eEF2 that form the ligand-binding pocket are oriented in a different manner relative to the rest of eEF2 compared with our previous structure of eEF2 in complex with the parent natural product sordarin. Yeast eEF2 is also shown to bind adenylic nucleotides, which can be displaced by sordarin, suggesting that ADP or ATP also bind to the three C-terminal domains of eEF2. Fusidic acid is a universal inhibitor of translation that targets EF-G or eEF2 and is widely used as an antibiotic against Gram-positive bacteria. Based on mutations conferring resistance to fusidic acid, cryo-EM reconstructions, and x-ray structures of eEF2, EF-G, and an EF-G homolog, we suggest that the conformation of EF-G stalled on the 70 S ribosome by fusidic acid is similar to that of eEF2 trapped on the 80 S ribosome by sordarin.

Roles and origins of leukocyte lipid bodies: proteomic and ultrastructural studies

Lipid bodies (LBs), multifunctional organelles present in most eukaryotic cells, are sites of eicosanoid formation in leukocytes; but little is known about the composition of leukocyte LBs or the biogenesis and internal structures of LBs from mammalian cells. Proteomic analyses of LBs purified from human monocytic U937 cells detected, common to LBs in other cells, proteins involved in cholesterol and triglyceride metabolism, Rab GTPases, and many membrane and endoplasmic reticulum (ER)-associated proteins. Newly lipid body (LB)-associated proteins included MRP-14, potentially involved in arachidonate transport, and ribosomal subunit proteins and translation regulatory proteins. Ultrastructurally, in U937 cells as well as human neutrophils and eosinophils, ribosomes are attached to and distributed within LBs, and LBs contain extensive ER-like membranes. The presence of ribosomes, ER-like membranes and many membrane-associated and ER luminal proteins within LBs, supports a new model by which enveloped ER-membranes and domains form LBs and indicates that LBs may be sites of protein synthesis.

Containing the Damage of Unfolded Proteins

The first stop for proteins bound for secretion from the cell or tethered to its membrane is the endoplasmic reticulum (ER). After secretory gene transcripts are translated into amino acid chains by ribosomes on the ER, they enter the ER’s labyrinthine network of membranes as extended polypeptide chains. Once the polypeptides are inside, ER enzymes assemble, modify, and fold them into their proper 3-D conformation in preparation for the next stop on the secretory pathway. When protein-folding demand exceeds ER capacity and clogs up the system with incorrectly folded proteins, sensors on the ER membrane stimulate the “unfolded protein response” (UPR). This ER-to-nucleus signaling pathway controls a vast gene-expression program that adjusts ER processing capacity and restores homeostasis by adjusting the size of the ER compartment, enlisting protein-folding enzymes, regulating the entry of amino acid chains, and removing irreparably folded proteins through the ER-associated protein degradation pathway. In a new study, Sebastian Bernales, Kent McDonald, and Peter Walter report a surprising link between another protein-degradation pathway, called autophagy, and the UPR. They show that as yeast cells expand their ER to accommodate increased demand, they also synthesize autophagosome-like bodies that sequester stacks of membrane from the expanded ER. This mechanism may allow the ER to keep the protein production line operating at a steady state even as misfolded proteins accumulate, and it may also control the size of this organelle. To characterize the UPR’s effect on ER structure and size, Bernales et al. chemically induced the UPR in yeast cells. Electron microscopy analysis revealed a “massive expansion” of the ER in treated cells compared with untreated cells. In yeast, the primary UPR sensor, Ire1, triggers the UPR by promoting the synthesis of the transcriptional activator Hac1. When the authors directly activated Ire1’s downstream transcriptional targets without causing protein misfolding (by artificially activating Hac1 production), they saw a similar increase. On closer inspection of UPR-induced cells, the authors were surprised to see an abundance of “autophagosome-like structures packed with tightly stacked membrane cisternae.” The structures were surrounded by double membranes like autophagosomes and were about the same size. The authors called them ER-containing autophagosomes (ERAs) after determining that their membrane contents came from the ER, based on the presence of ribosomes and other ER proteins and extensive membrane visualization analysis. Three hours after UPR induction, the majority of cells had proliferated ERs and about 20% of cells had ERAs. None of the ERA-containing cells had an expanded ER, suggesting that ERAs provide a means to downsize the ER to counteract expansion. Given the ERAs’ structural resemblance to autophagosomes, the authors reasoned that they may function similarly as well. In autophagy, cells recycle excess or worn out organelles (and sections of cytoplasm) by packaging them into autophagosomes that fuse with vesicles (vacuoles or lysosomes) equipped with protein-digesting acid hydrolases. Starvation induces macro-autophagy, which indiscriminately cannibalizes cytoplasm sections to release nutrients from degraded molecules. By contrast, ERAs are highly selective in sequestering ER membranes. To investigate ERA function, the authors tracked the fate of an early component of autophagosome formation, Atg8, using a fluorescent version of the protein. In nitrogen-starved yeast, Atg8 concentrates in pre-autophagosomal structures (PASs) near the vacuole, where Atg8’s fluorescent domain is removed. It is thought that PASs stimulate the formation of autophagosomes, which then fuse with the vacuole and dump their contents. As expected, nitrogen starvation upregulated Atg8 (but not the UPR relay signal). Chemically induced UPR also increased Atg8 production, but did not cleave Atg8’s fluorescent domain, indicating that Atg8’s fate differs under these different physiological stresses. But more surprising, this result identifies the autophagy gene ATG8 as a UPR target. The authors go on to show that UPR induction produces a “vast proliferation” of Atg8-containing PASs, located near vacuoles or ERAs (when cells had them). Since no ERAs formed in cells lacking ATG8, the gene appears to play a part in ERA formation. ATG8, along with five other autophagy genes, is also required for cell growth under UPR-inducing conditions. Altogether, these results establish a surprising link between autophagy and the UPR. Autophagy, the authors conclude, supplements the UPR by selectively “self-eating” excess ER to help cells weather the potentially fatal consequences of ER stress—in contrast to starvation-induced autophagy, which cannibalizes cellular contents for their metabolites. Intriguingly, autophagy genes can be enlisted by Ire1 but also independently of the Ire1-dependent UPR pathway, thus providing a rich platform for exploring alternative ER-to-nucleus signaling pathways in yeast. Future studies can begin to characterize the molecular mechanisms required for ERA formation and more fully explore the cellular functions of “ER-phagy.”

Molecular dissection of the mycobacterial stringent response protein Rel.

Latency in Mycobacterium tuberculosis poses a barrier in its complete eradication. Overexpression of certain genes is one of the factors that help these bacilli survive inside the host during latency. Among these genes, rel, which leads to the expression of Rel protein, plays an important role by synthesizing the signaling molecule ppGpp using GDP and ATP as substrates, thereby changing bacterial physiology. In Gram-negative bacteria, the protein is thought to be activated in vivo in the presence of ribosome by sensing uncharged tRNA. In the present report, we show that Rel protein from Mycobacterium smegmatis, which is highly homologous to M. tuberculosis Rel, is functional even in the absence of ribosome and uncharged tRNA. From the experiments presented here, it appears that the activity of Rel(Msm) is regulated by the domains present at the C terminus, as the deletion of these domains results in higher synthesis activity, with little change in hydrolysis of ppGpp. However, in the presence of tRNA, though the synthesis activity of the full-length protein increases to a certain extent, the hydrolysis activity undergoes drastic reduction. Full-length Rel undergoes multimerization involving interchain disulfide bonds. The synthesis of pppGpp by the full-length protein is enhanced in the reduced environment in vitro, whereas the hydrolysis activity does not change significantly. Mutations of cysteines to serines result in monomerization with a simultaneous increase in the synthesis activity. Finally, it has been possible to identify the unique cysteine, of six present in Rel, required for tRNA-mediated synthesis of ppGpp.

Growth cones make proteins, too

Axons branch out from neurons as they respond to chemical cues in their extracellular environment. Until recently, many scientists did not believe that elongating axons could synthesize proteins locally. But in the past three years, this view has been largely overturned. One of the first clues that protein synthesis might occur in the growing tips of axons—the growth cones—came from morphological studies conducted over 30 years ago by Virginia Tennyson. In the mid-1960s, several electron microscopy studies of neurons had been published, but few of them focused on the growth cone. Tennyson, then a researcher at Columbia University, decided to examine the axons of fetal rabbit dorsal root neuroblasts at 11–12 days, a time in development when many growth cones are present. “I remember, I wanted to study growth cones,” recalls Tennyson. “I certainly was not expecting to see any ribosomes.” Tennyson observed clusters of particles along the length of an entire axon and in several growth cones. The particles were 150–250 A in diameter and morphologically identical to ribosomes (Tennyson, 1970). “The presence of ribosomes in the early embryonic axons suggests that protein synthesis may continue in these segments at a considerable distance from the perikaryon [neuron cell body],” Tennyson wrote in her 1970 paper. “Of course I had no evidence of protein synthesis at that time, so I did not want to make too much of that observation,” she says. Shortly after Tennyson's study, further ultrastructural analyses confirmed the presence of polyribosomes in growth cones of cultured neurons (Yamada et al., 1971; Bunge, 1973). Figure Ribosomes (arrows) turned up in axons. Since then, several studies have documented ribosomes, mRNA, translational initiation proteins, and protein synthesis in axons and growth cones. Douglas Campbell and Christine Holt (University of Cambridge) demonstrated that molecules that guide the growth of axons rapidly trigger protein synthesis in isolated retinal growth cones (Campbell and Holt, 2001). Inhibition of protein synthesis by translation blockers abolishes the response of these growth cones to guidance molecules. This and other studies (Brittis et al., 2002; Zheng et al., 2001) showed that, at least in vitro, fast, local synthesis of proteins not only occurs but is necessary for guiding axon growth in response to external cues. “In retrospect,” muses Holt, “it is surprising how remarkable everyone thought this was.” Brittis, P.A., et al. 2002. Cell. 110:223–235. [PubMed] Bunge, M.B. 1973. J. Cell Biol. 56:713–735. [PubMed] Campbell, D.S., and C.E. Holt. 2001. Neuron. 32:1013–1026. [PubMed] Tennyson, V.M. 1970. J. Cell Biol. 44:62–79. [PubMed] Yamada, K.M., et al. 1971. J. Cell Biol. 49:614–635. [PubMed] Zheng, J.Q., et al. 2001. J. Neurosci. 21:9291–9303. [PubMed]

Prokaryotic cells of the deep sub-seafloor biosphere identified as living bacteria

Chemical analyses of the pore waters from hundreds of deep ocean sediment cores have over decades provided evidence for ongoing processes that require biological catalysis by prokaryotes. This sub-seafloor activity of microorganisms may influence the surface Earth by changing the chemistry of the ocean and by triggering the emission of methane, with consequences for the marine carbon cycle and even the global climate. Despite the fact that only about 1% of the total marine primary production of organic carbon is available for deep-sea microorganisms, sub-seafloor sediments harbour over half of all prokaryotic cells on Earth. This estimation has been calculated from numerous microscopic cell counts in sediment cores of the Ocean Drilling Program. Because these counts cannot differentiate between dead and alive cells, the population size of living microorganisms is unknown. Here, using ribosomal RNA as a target for the technique known as catalysed reporter deposition-fluorescence in situ hybridization (CARD-FISH), we provide direct quantification of live cells as defined by the presence of ribosomes. We show that a large fraction of the sub-seafloor prokaryotes is alive, even in very old (16 million yr) and deep (> 400 m) sediments. All detectable living cells belong to the Bacteria and have turnover times of 0.25-22 yr, comparable to surface sediments.

Відновлення активності аміноацил-тРНК синтетаз вищих еукаріотів та їхня стабілізація у присутності рибосом

Відновлення активності аміноацил-тРНК синтетаз вищих еукаріотів та їхня стабілізація у присутності рибосом

Structural Insights into the Stringent Response

The stringent response allows bacteria to survive conditions of amino acid starvation through regulation of transcription by derivatives of guanosine triphosphate or guanosine diphosphate collectively called alarmone or ppGpp [for guanosine 3′-diphosphate 5′-triphosphate or guanosine 3′,5′-bis(diphosphate)]. Alarmone interacts with RNA polymerase (RNAP) to control its activity in a promoter-selective mechanism. Two groups report structural analysis of proteins involved in this stringent response. Hogg et al . analyzed the structure of the enzyme Rel Seq , which is responsible for both the production and degradation of ppGpp, from the Streptococcus dysgalactiae subspecies e quisimilis . The structure indicated that the two active sites are 30 Å apart. Two conformations of the protein were observed and binding of an unusual ppGpp derivative, guanosine 5′-diphosphate-2′:3′-cyclic monophosphate (ppG2′:3′p), to the hydrolase site appeared to lock the protein into a hydrolase-on and synthetase-off conformation. In the synthetase-on structure, GDP binding to a catalytically competent synthetase site coincided with a nonproductive, unliganded state at the hydrolase site. The protein crystallized lacked the ribosome-binding portion, which is responsible for regulating the balance of the catalytic activities in response to the presence of ribosome with nonacylated tRNAs. However, the structure suggests an allosteric mechanism for deactivating synthetase activity in response to conformational changes induced by nucleotide binding to the hydrolase domain. A second paper by Artsimovitch et al. that explored the structure of the Thermus thermophilus RNAP homoenzyme with ppGpp provided insight into how ppGpp can stimulate transcription at some promoters and inhibit transcription at others. The crystal contained two RNAP molecules, which appeared to bind ppGpp in alternate orientations. The two RNAP molecules were designated RNAP1, in which ppGpp was bound such that the 5′ diphosphate of ppGpp was closest to the active site Asp residues, and RNAP2, in which the ppGpp bound such that the 3′ diphosphate of ppGpp was closest to the active site Asp residues. These different ppGpp binding orientations produce differences between RNAP1 and RNAP2 in their active site conformations and in the conformation of subunits that interacted with the DNA promoter in the open complex. The RNAP1 conformation may have increased catalytic activity due to changes in the affinity of the active site for Mg 2+ ions, whereas the activity of RNAP in the RNAP2 conformation may be inhibited because binding of the second Mg 2+ ion is unfavorable. In addition, the RNAP2 conformation may allow ppGpp to bind to cytosines in the nontemplate DNA strand, thereby destabilizing the DNA open complex and inhibiting transcription. Thus, these two orientations of ppGpp may produce conformations of RNAP that have different activities. The interaction with the nontemplate DNA may contribute to the promoter-selective effects of ppGpp. T. Hogg, U. Mechold, H. Malke, M. Cashel, R. Hilgenfeld, Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response. Cell 117 , 57-68 (2004). [Online Journal] I. Artsimovitch, V. Patlan, S.-i. Sekine, M. N. Vassylyeva, T. Hosaka, K. Ochi, S. Yokoyama, D. G. Vassylyev, Structural basis for transcription regulation by alarmone ppGpp. Cell 117 , 299-310 (2004). [Online Journal]

Messenger RNA profiling of human platelets by microarray hybridization

Platelets are generally believed to be inactive in terms of de novo protein synthesis. On the other hand, the presence of ribosomes and mRNA molecules is well established. Many studies have used reverse transcriptase (RT) -PCR for detection of gene transcripts in platelets. As RT-PCR is a very sensitive method, any leukocyte contamination of platelet preparations can lead to false results. We performed three filtration procedures to minimize leukocyte contamination of pooled buffy-coat platelet concentrates prior to RNA isolation. Furthermore, by applying a genomic PCR approach with 50 amplification cycles we demonstrated that nucleated cells were not detectable. Microarray hybridization was used to analyze 9,850 individual human genes in RNA from purified platelets. In total we identified 1,526 (15.5%) positive genes. The data were confirmed in six individual experiments each performed on a PC pooled from four individual blood donations. Genes specific for nucleated blood cells such as CD4, CD83 and others were negative and verified the purity of PC. Overrepresentation of positive genes was found in the functional categories of glycoproteins/integrins (22.6% vs. 15.5%, p=0.029) and receptors (20.7% vs. 15.5%, p<0.001). Gene transcripts encoding RANTES, GRO-alpha, MIP-1alpha, MIP-1beta, and others were found at high levels of signal intensity and confirmed literature data. This work provides a mRNA profile of human platelets and a complete list of results can be downloaded from the website of our institute www.ma.uni-heidelberg.de/inst/iti/plt_array.xls. The knowledge about gene transcripts may have an impact on the characterization of novel proteins and their functions in platelets.

SRP-dependent co-translational targeting and SecA-dependent translocation analyzed as individual steps in the export of a bacterial protein.

Recently it has been recognized that the signal recognition particle (SRP) of Escherichia coli represents a specific targeting device for hydrophobic inner membrane proteins. It has remained unclear, however, whether the bacterial SRP functions in concert with SecA, which is required for the translocation of secretory proteins across the inner membrane. Here, we have analyzed a hybrid protein constructed by fusing the signal anchor sequence of an SRP-dependent inner membrane protein (MtlA) to the mature part of an exclusively SecA-requiring secretory protein (OmpA). We show that the signal anchor sequence of MtlA confers the novel properties onto nascent chains of OmpA of being co-translationally recognized and targeted to SecY by SRP. Once targeted to SecY, ribosome-associated nascent chains of the hybrid protein, however, remain untranslocated unless SecA is present. These results indicate that SRP and SecA cooperate in a sequential, non-overlapping manner in the topogenesis of those membrane proteins which, in addition to a signal anchor sequence, harbor a substantial hydrophilic domain to be translocated into the periplasm.

Ribosome-mediated Folding of Partially Unfolded Ricin A-chain

After endocytic uptake by mammalian cells, the cytotoxic protein ricin is transported to the endoplasmic reticulum, whereupon the A-chain must cross the lumenal membrane to reach its ribosomal substrates. It is assumed that membrane traversal is preceded by unfolding of ricin A-chain, followed by refolding in the cytosol to generate the native, biologically active toxin. Here we describe biochemical and biophysical analyses of the unfolding of ricin A-chain and its refolding in vitro. We show that native ricin A-chain is surprisingly unstable at pH 7.0, unfolding non-cooperatively above 37 degrees C to generate a partially unfolded state. This species has conformational properties typical of a molten globule, and cannot be refolded to the native state by manipulation of the buffer conditions or by the addition of a stem-loop dodecaribonucleotide or deproteinized Escherichia coli ribosomal RNA, both of which are substrates for ricin A-chain. By contrast, in the presence of salt-washed ribosomes, partially unfolded ricin A-chain regains full catalytic activity. The data suggest that the conformational stability of ricin A-chain is ideally poised for translocation from the endoplasmic reticulum. Within the cytosol, ricin A-chain molecules may then refold in the presence of ribosomes, resulting in ribosome depurination and cell death.

Identification of elongation factor 2 as the essential protein targeted by sordarins in Candida albicans.

The target for sordarins in Candida albicans has been elucidated. Kinetic experiments of sordarin inhibition as well as displacement experiments showed that the formation of a sordarin-target complex follows a reversible mechanism. Binding of tritiated drug to the target is enhanced in the presence of ribosomes. Isolation of the target by classical protein purification methods has allowed us to identify it as elongation factor 2. This is in agreement with the nature of sordarin derivatives as specific inhibitors of the elongation cycle within protein synthesis in yeasts.

Selenocysteine inserting RNA elements modulate GTP hydrolysis of elongation factor SelB.

Elongation factor SelB is required for the incorporation of the amino acid selenocysteine into proteins in Escherichia coli. Selenocysteine incorporation is thought to be achieved by simultaneous binding of SelB to selenocysteyl-tRNASec and to an mRNA hairpin structure located 3' adjacent to the UGA selenocysteine codon. SelB was shown previously to bind to GTP or GDP in a molar ratio of 1:1. Here, we demonstrate that SelB, like EF-Tu, exhibits a low intrinsic GTPase activity in the absence of ribosomes. As shown for EF-Tu, GTPase activity of SelB is stimulated by the presence of E. coli 70S ribosomes; the apparent K(m) for GTP hydrolysis is 55 microM. Interestingly, in the presence of the mRNA hairpin which promotes selenocysteine incorporation, GTPase activity of SelB increases additionally by 3-4-fold; stimulation is due to kcat increasing from 0.05/min in the absence to 0.16/min in the presence of the mRNA hairpin. This mRNA-induced stimulation of SelB GTPase activity depends on the presence of ribosomes. The minimal region of the mRNA hairpin capable to stimulate GTP hydrolysis by SelB locates within the upper half of the hairpin; this part of the mRNA structure was demonstrated previously to be sufficient for binding of the mRNA to SelB. On the basis of these results, we propose that binding of the mRNA hairpin to SelB induces a conformational switch within SelB thereby promoting an increase in ribosome-mediated GTP hydrolysis.

Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent 'envelope smoothing' event.

We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying ('rough') vesicles, many of which bound directly to chromatin, and 'smooth' vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatin-attachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed 'envelope smoothing' occurred 5-15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that 'smoothing' required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.