Transmembrane segments of nascent polytopic membrane proteins control cytosol/ER targeting during membrane integration

During cotranslational integration of a eukaryotic multispanning polytopic membrane protein (PMP), its hydrophilic loops are alternately directed to opposite sides of the ER membrane. Exposure of fluorescently labeled nascent PMP to the cytosol or ER lumen was detected by collisional quenching of its fluorescence by iodide ions localized in the cytosol or lumen. PMP loop exposure to the cytosol or lumen was controlled by structural rearrangements in the ribosome, translocon, and associated proteins that occurred soon after a nascent chain transmembrane segment (TMS) entered the ribosomal tunnel. Each successive TMS, although varying in length, sequence, hydrophobicity, and orientation, reversed the structural changes elicited by its predecessor, irrespective of loop size. Fluorescence lifetime data revealed that TMSs occupied a more nonpolar environment than secretory proteins inside the aqueous ribosome tunnel, which suggests that TMS recognition by the ribosome involves hydrophobic interactions. Importantly, the TMS-triggered structural rearrangements that cycle nascent chain exposure between cytosolic and lumenal occur without compromising the permeability barrier of the ER membrane.     

Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins

Fluorescence resonance energy transfer measurements reveal that a transmembrane sequence within a nascent membrane protein folds into a compact conformation near the peptidyltransferase center and remains folded as the sequence moves through a membrane bound ribosome into the translocon. This compact conformation is compatible with an alpha helix because nearly the same energy transfer efficiency was observed when the transmembrane sequence was integrated into the lipid bilayer. Since the transmembrane sequence unfolds upon emerging from a free ribosome, this nascent chain folding is ribosome induced and stabilized. In contrast, a nascent secretory protein is in an extended conformation in the exit tunnel. Furthermore, two ribosomal proteins photo-crosslink to nascent membrane but not secretory proteins. These interactions coincide with the previously described sequential closing and opening of the two ends of the aqueous translocon pore, thereby suggesting that ribosomal recognition of nascent chain folding controls the operational mode of the translocon at the ER membrane.     

The structural and functional coupling of two molecular machines,the ribosome and the translocon

Ribosomes synthesizing secretory and membrane proteins are bound to translocons at the membrane of the endoplasmic reticulum (ER). Both the ribosome and translocon are complex macromolecular machines whose structural and functional interactions are poorly understood. A new study by Pool (Pool, M.R. 2009. J. Cell Biol. 185:889–902) has now shown that the structure of the translocon is dictated by the identity of the protein being synthesized by the ribosome, thereby demonstrating that the two macromolecular machines are structurally coupled for functional purposes. The study also identifies an unexpected component in the apparent molecular linkage that connects the two machines, a discovery that shows the current view of translocon structure is oversimplified.The mammalian translocon is assembled from four core proteins (the heterotrimeric Sec61α, Sec61β, Sec61γ, and translocating nascent chain–associated membrane protein [TRAM]) and various accessory proteins, only some of which have well-defined functions (e.g., the signal peptidase and the oligosaccharyltransferase; Rapoport, 2007). This assembly forms the aqueous, gated pore that transports nascent proteins through the membrane (Crowley et al., 1994). Because two molecular machines, the ribosome and the translocon, function simultaneously on the same nascent protein during cotranslational protein trafficking, they presumably function together as a unit (the ribosome–translocon complex [RTC]) that includes all proteins regularly associated with the translocon. Yet these machines are commonly treated as separate entities in papers and talks; ribosomologists tend to assume that membrane-bound ribosomes are indistinguishable from free ribosomes except for their location, whereas translocophiles tend to consider the ribosome simply a source of substrate that docks on the translocon. However, the results published in this issue (see Pool on p. 889) demonstrate that the identity of the nascent chain being synthesized alters RTC structure, thereby revealing that the two machines are indeed coupled.The nascent chain moves through the large ribosomal subunit via an ∼100-Å-long tunnel that is contiguous with the aqueous pore formed by the translocon (Fig. 1 A; Crowley et al., 1994; Beckmann et al., 1997; Nissen et al., 2000). Nascent chain control of protein trafficking from inside the ribosome was first identified when the location of a nascent chain transmembrane segment (TMS) in the tunnel was found to dictate whether the nascent chain was exposed to the cytosolic, lumenal, or neither side of the ER membrane (Liao et al., 1997). It was postulated that a weakly nonpolar patch in the tunnel nucleated the folding of the TMS into an α-helix, which in turn elicited conformational changes in the RTC that triggered complementary changes at each end of the pore to minimize ion passage/leakage through the translocon during integration. A later study revealed that ribosome-induced folding of a nascent chain TMS did occur, and this folding coincided with TMS photocrosslinking to Rpl17 at a constriction in the tunnel (Fig. 1 B; Woolhead et al., 2004), a site formed in part by a loop of Rpl17 that extends far into the large ribosomal subunit (Nissen et al., 2000; Berisio et al., 2003).Open in a separate windowFigure 1.Nascent chain control of translocon structure from inside the ribosome. (A and B) A nascent secretory protein is fully extended during synthesis (A), whereas the TMS in a nascent membrane protein (B) folds into an α-helix upon reaching a tunnel constriction formed by Rpl4 and Rpl17. Although Sec61β is always adjacent to Rpl17 in an RTC, RAMP4 is recruited to the RTC and is cross-linked to Rpl17 only when a TMS reaches the constriction. The cross-linking of Rpl17 to a TMS and to RAMP4 coincides with the BiP-mediated closure (either directly, as depicted, or indirectly) of the lumenal end of the aqueous pore and the subsequent opening of the ion-tight ribosome–translocon junction (depicted by a tilting of the ribosomal subunit). PTC, peptidyl transferase center.Pool (2009) found that ribosomal protein Rpl17, located primarily at the ribosomal surface near the tunnel exit, was chemically cross-linked to Sec61β, thereby showing that Sec61β is adjacent to Rpl17 in all RTCs. But when the ribosome was synthesizing a membrane protein, Rpl17 also cross-linked to RAMP4, a small ribosome-associated membrane protein associated with the translocon (Schröder et al., 1999). Strikingly, Rpl17 cross-linking to RAMP4 was detected only after the TMS in the nascent chain reached the tunnel constriction (Fig. 1 B). The coincidence of these two cross-linking events, TMS to Rpl17 and Rpl17 to RAMP4, indicates that direct contact between the nascent chain TMS and Rpl17 inside the tunnel triggered a conformational change that was transmitted through the Rpl17 extension to the ribosomal surface and that stimulated RAMP4 association with the RTC close to Rpl17. Thus, a structural feature found only in nascent membrane proteins caused a structural realignment of the RTC. This change correlates with the transition of RTC operations from translocation to integration seen in earlier studies (Liao et al., 1997; Haigh and Johnson, 2002; Woolhead et al., 2004).The data shown by Pool (2009) also demonstrate that the translocon, at least in mammals, is not as structurally defined as has been portrayed. Uncertainties in the composition, stoichiometry, exchangeability, and macromolecular arrangement of the core and associated translocon proteins, as well as translocon dynamics and homogeneity, have been recognized for some time (Johnson and van Waes, 1999), but only recently have translocon heterogeneity (Snapp et al., 2004; Shibatani et al., 2005), conformational changes (Hamman et al., 1997), and exchangeable membrane proteins (e.g., importin α-16 appears to be a sorting factor for inner nuclear membrane proteins; Saksena et al., 2004, 2006) been documented. The data now show that RAMP4 in the bilayer associates with the RTC near Rpl17 when a TMS binds Rpl17 inside the ribosome Pool, 2009). Thus, translocon structure is dynamic and involves more proteins than the core Sec61α, Sec61β, Sec61γ, and TRAM proteins. Equally important, as shown by Pool (2009), translocon structure can be altered by a nascent chain structural feature from inside the ribosome. Thus, the structures of the ribosome and translocon are intimately coupled, and the conversion of the RTC from one functional state to another involves structural changes (e.g., the introduction of a new protein) not accommodated in current models of translocon structure and function.The study by Pool (2009) also emphasizes the importance of using multiple approaches to examine complex systems, especially those containing membranes. Protein crystallography and cryo-EM have made tremendous strides recently in describing the structures of molecular assemblies such as the ribosome and translocon (Beckmann et al., 1997; Nissen et al., 2000; Berisio et al., 2003; Van den Berg et al., 2004), and it is difficult to overstate how important those studies have been in terms of influencing subsequent RTC research. Yet no single technique is a panacea, and the data shown by Pool (2009) dramatically highlight the limitations of assuming that crystallographic and cryo-EM studies provide all that one needs to understand the structural aspects of how a machine works, much less the functional, mechanistic, and/or regulatory aspects. For example, RAMP4, the core protein TRAM, and other translocon-associated proteins have not been identified in cryo-EM images of the detergent-solubilized RTC as a result of their small size, flexibility, and/or weak association with the Sec61 core. Similarly, the existence of ribosome-induced TMS folding inside the ribosome tunnel and its regulation of nascent chain accessibility to alternate sides of the ER membrane would not have been detected with samples that had been detergent-treated and lacked water, the lipid bilayer, and some of the core and associated translocon proteins. Thus, although models derived from crystallographic and cryo-EM images of detergent-treated, incomplete, anhydrous, and nonfunctional RTC samples may prove to be accurate, well-designed experiments and controls using intact samples in aqueous solution must be performed to directly correlate structural and functional states and thereby fully appreciate subtle and sophisticated operational and regulatory mechanisms that do not withstand harsh treatment.There is much left to learn about the RTC. Rpl17 acts as a direct communication conduit between a TMS in the tunnel and the translocon, but it remains to be seen whether RAMP4 then mediates transmembrane communication and triggers BiP binding. Similarly, RAMP4 interactions with the RTC have not been characterized nor have the protein composition, arrangement, and dynamics of the mammalian ER translocon in intact membranes. Thus, additional intriguing surprises are ahead.     

Importin-alpha-16 is a translocon-associated protein involved in sorting membrane proteins to the nuclear envelope

A viral inner nuclear membrane-sorting motif sequence (INM-SM) was used to identify proteins that recognize integral membrane proteins destined for the INM. Herein we describe importin-alpha-16, a membrane-associated isoform of Spodoptera frugiperda importin-alpha that contains the C-terminal amino acid residues comprising armadillo helical-repeat domains 7-10. In the endoplasmic reticulum (ER) membrane, importin-alpha-16 is adjacent to the translocon protein Sec61alpha. Importin-alpha-16 cross-links to the INM-SM sequence as it emerges from the ribosomal tunnel and remains adjacent to the INM-SM after INM-SM integration into the ER membrane and release from the translocon. Cross-linking results suggest that importin-alpha-16 discriminates between INM- and non-INM-directed proteins. Thus, it seems that during and after cotranslational membrane integration, importin-alpha-16 is involved in the trafficking of integral membrane proteins to the INM.     

A new role for BiP: closing the aqueous translocon pore during protein integration into the ER membrane.

In mammalian cells, most membrane proteins are inserted cotranslationally into the ER membrane at sites termed translocons. Although each translocon forms an aqueous pore, the permeability barrier of the membrane is maintained during integration, even when the otherwise tight ribosome-translocon seal is opened to allow the cytoplasmic domain of a nascent protein to enter the cytosol. To identify the mechanism by which membrane integrity is preserved, nascent chain exposure to each side of the membrane was determined at different stages of integration by collisional quenching of a fluorescent probe in the nascent chain. Comparing integration intermediates prepared with intact, empty, or BiP-loaded microsomes revealed that the lumenal end of the translocon pore is closed by BiP in an ATP-dependent process before the opening of the cytoplasmic ribosome-translocon seal during integration. This BiP function is distinct from its previously identified role in closing ribosome-free, empty translocons because of the presence of the ribosome at the translocon and the nascent membrane protein that extends through the translocon pore and into the lumen during integration. Therefore, BiP is a key component in a sophisticated mechanism that selectively closes the lumenal end of some, but not all, translocons occupied by a nascent chain. By using collisional quenchers of different sizes, the large internal diameter of the ribosome-bound aqueous translocon pore was found to contract when BiP was required to seal the pore during integration. Therefore, closure of the pore involves substantial conformational changes in the translocon that are coupled to a complex sequence of structural rearrangements on both sides of the ER membrane involving the ribosome and BiP.     

Structural insight into the role of the ribosomal tunnel in cellular regulation

Nascent proteins emerge out of ribosomes through an exit tunnel, which was assumed to be a firmly built passive path. Recent biochemical results, however, indicate that the tunnel plays an active role in sequence-specific gating of nascent chains and in responding to cellular signals. Consistently, modulation of the tunnel shape, caused by the binding of the semi-synthetic macrolide troleandomycin to the large ribosomal subunit from Deinococcus radiodurans, was revealed crystallographically. The results provide insights into the tunnel dynamics at high resolution. Here we show that, in addition to the typical steric blockage of the ribosomal tunnel by macrolides, troleandomycin induces a conformational rearrangement in a wall constituent, protein L22, flipping the tip of its highly conserved beta-hairpin across the tunnel. On the basis of mutations that alleviate elongation arrest, the tunnel motion could be correlated with sequence discrimination and gating, suggesting that specific arrest motifs within nascent chain sequences may induce a similar gating mechanism.     

The ribosomal tunnel as a functional environment for nascent polypeptide folding and translational stalling

As the nascent polypeptide chain is being synthesized, it passes through a tunnel within the large ribosomal subunit and emerges at the solvent side where protein folding occurs. Despite the universality and conservation of dimensions of the ribosomal tunnel, a functional role for the ribosomal tunnel is only beginning to emerge: Rather than a passive conduit for the nascent chain, accumulating evidence indicates that the tunnel plays a more active role. In this article, we discuss recent structural insights into the role of the tunnel environment, and its implications for protein folding, co-translational targeting and translation regulation.     

A cradle for new proteins: trigger factor at the ribosome

Newly synthesized proteins leave the ribosome through a narrow tunnel in the large subunit. During ongoing synthesis, nascent protein chains are particularly sensitive to aggregation and degradation because they emerge from the ribosome in an unfolded state. In bacteria, the first protein to interact with nascent chains and facilitate their folding is the ribosome-associated chaperone trigger factor. Recently, crystal structures of trigger factor and of its ribosome-binding domain in complex with the large ribosomal subunit revealed that the chaperone adopts an extended 'dragon-shaped' fold with a large hydrophobic cradle, which arches over the exit of the ribosomal tunnel and shields newly synthesized proteins. These structural results, together with recent biochemical data on trigger factor and its interplay with other chaperones and factors that interact with the nascent chain, provide a comprehensive view of the role of trigger factor during co-translational protein folding.     

A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translation

The immediate environment of nascent membrane proteins undergoing integration into the ER membrane was investigated by photocrosslinking. Nascent polypeptides of different lengths, each containing a single IgM transmembrane sequence that functions either as a stop-transfer or a signal-anchor sequence, were synthesized by in vitro translation of truncated mRNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes. This yielded nascent chains with photoreactive probes at one end of the transmembrane sequence where two lysine residues are located. When irradiated, these nascent chains reacted covalently with several ER proteins. One prominent crosslinking target was a glycoprotein similar in size to a protein termed mp39, shown previously to be situated adjacent to a secretory protein during its translocation across the ER membrane (Krieg, U. C., A. E. Johnson, and P. Walter. 1989. J. Cell Biol. 109:2033-2043; Wiedmann, M., D. Goerlich, E. Hartmann, T. V. Kurzchalia, and T. A. Rapoport. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 257:263-268) and likely to be identical to a protein previously designated the signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature (Lond.). 328:830-833). Changing the orientation of the transmembrane domain in the bilayer, or making the transmembrane domain the first topogenic sequence in the nascent chain instead of the second, did not significantly alter the identities of the ER proteins that were the primary crosslinking targets. Furthermore, the nascent chains crosslinked to the mp39-like glycoprotein and other microsomal proteins even after the cytoplasmic tail of the nascent chain had been lengthened by nearly 100 amino acids beyond the stop-transfer sequence. Yet when the nascent chain was allowed to terminate normally, the major photocrosslinks were no longer observed, including in particular that to the mp39-like glycoprotein. These results show that the transmembrane segment of a nascent membrane protein is located adjacent to the mp39-like glycoprotein and other ER proteins during the integration process, and that at least a portion of the nascent chain remains in close proximity to these ER proteins until translation has been completed.     

Protein translocation across the endoplasmic reticulum membrane: identification by photocross-linking of a 39-kD integral membrane glycoprotein as part of a putative translocation tunnel

The molecular environment of secretory proteins during translocation across the ER membrane was examined by photocross-linking. Nascent preprolactin chains of various lengths, synthesized by in vitro translation of truncated messenger RNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes, were used to position photoreactive probes at various locations within the membrane. Upon photolysis, each nascent chain species was cross-linked to an integral membrane glycoprotein with a deduced mass of 39 kD (mp39) via photoreactive lysines located in either the signal sequence or the mature prolactin sequence. Thus, different portions of the nascent preprolactin chain are in close proximity to the same membrane protein during the course of translocation, and mp39 therefore appears to be part of the translocon, the specific site of protein translocation across the ER membrane. The similarity of the molecular and cross-linking properties of mp39 and the glyco-protein previously identified as a signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature [Lond.]. 328: 830-833) suggests that these two proteins may be identical. Our data indicate, however, that mp39 does not (or not only) function as a signal sequence receptor, but rather may be part of a putative translocation tunnel.     

N-linked glycan recognition and processing: the molecular basis of endoplasmic reticulum quality control

Nascent polypeptides emerging into the lumen of the endoplasmic reticulum (ER) are N-glycosylated on asparagines in Asn-Xxx-Ser/Thr motifs. Processing of the core oligosaccharide eventually determines the fate of the associated polypeptide by regulating entry into and retention by the calnexin chaperone system, or extraction from the ER folding environment for disposal. Recent advances have shown that at least two N-glycans are necessary for protein access to the calnexin chaperone system and that polypeptide cycling in the system is a rather rare event, which, for folding-defective polypeptides, is activated only upon persistent misfolding. Additionally, dismantling of the polypeptide-bound N-glycan interrupts futile folding attempts, and elicits preparation of the misfolded chain for dislocation into the cytosol and degradation.     

NAC functions as a modulator of SRP during the early steps of protein targeting to the endoplasmic reticulum

Nascent polypeptide-associated complex (NAC) was initially found to bind to any segment of the nascent chain except signal sequences. In this way, NAC is believed to prevent mistargeting due to binding of signal recognition particle (SRP) to signalless ribosome nascent chain complexes (RNCs). Here we revisit the interplay between NAC and SRP. NAC does not affect SRP function with respect to signalless RNCs; however, NAC does affect SRP function with respect to RNCs targeted to the endoplasmic reticulum (ER). First, early recruitment of SRP to RNCs containing a signal sequence within the ribosomal tunnel is NAC dependent. Second, NAC is able to directly and tightly bind to nascent signal sequences. Third, SRP initially displaces NAC from RNCs; however, when the signal sequence emerges further, trimeric NAC·RNC·SRP complexes form. Fourth, upon docking to the ER membrane NAC remains bound to RNCs, allowing NAC to shield cytosolically exposed nascent chain domains not only before but also during cotranslational translocation. The combined data indicate a functional interplay between NAC and SRP on ER-targeted RNCs, which is based on the ability of the two complexes to bind simultaneously to distinct segments of a single nascent chain.     

Folding at the birth of the nascent chain: coordinating translation with co-translational folding

In the living cells, the folding of many proteins is largely believed to begin co-translationally, during their biosynthesis at the ribosomes. In the ribosomal tunnel, the nascent peptide may establish local interactions and stabilize α-helical structures. Long-range contacts are more likely outside the ribosomes after release of larger segments of the nascent chain. Examples suggest that domains can attain native-like structure on the ribosome with and without population of folding intermediates. The co-translational folding is limited by the speed of the gradual extrusion of the nascent peptide which imposes conformational restraints on its folding landscape. Recent experimental and in silico modeling studies indicate that translation kinetics fine-tunes co-translational folding by providing a time delay for sequential folding of distinct portions of the nascent chain.     

Molecular mechanism and structure of Trigger Factor bound to the translating ribosome

Ribosome-associated chaperone Trigger Factor (TF) initiates folding of newly synthesized proteins in bacteria. Here, we pinpoint by site-specific crosslinking the sequence of molecular interactions of Escherichia coli TF and nascent chains during translation. Furthermore, we provide the first full-length structure of TF associated with ribosome-nascent chain complexes by using cryo-electron microscopy. In its active state, TF arches over the ribosomal exit tunnel accepting nascent chains in a protective void. The growing nascent chain initially follows a predefined path through the entire interior of TF in an unfolded conformation, and even after folding into a domain it remains accommodated inside the protective cavity of ribosome-bound TF. The adaptability to accept nascent chains of different length and folding states may explain how TF is able to assist co-translational folding of all kinds of nascent polypeptides during ongoing synthesis. Moreover, we suggest a model of how TF's chaperoning function can be coordinated with the co-translational processing and membrane targeting of nascent polypeptides by other ribosome-associated factors.     

Localization of the trigger factor binding site on the ribosomal 50S subunit

In Escherichia coli, protein folding is undertaken by three distinct sets of chaperones, the DnaK-DnaJ and GroEL-GroES systems and the trigger factor (TF). TF has been proposed to be the first chaperone to interact with the nascent polypeptide chain as it emerges from the tunnel of the 70S ribosome and thus probably plays an important role in co-translational protein folding. We have made complexes with deuterated ribosomes (50S subunits and 70S ribosomes) and protated TF and determined the TF binding site on the respective complexes using the neutron scattering technique of spin-contrast variation. Our data suggest that the TF binds in the form of a homodimer. On both the 50S subunit and the 70S ribosome, the TF position is in proximity to the tunnel exit site, near ribosomal proteins L23 and L29, located on the back of the 50S subunit. The positions deviate from one another, such that the position on the 70S ribosome is located slightly further from the tunnel than that determined for the 50S subunit alone. Nevertheless, from both determined positions interaction between TF and a short nascent chain of 57 amino acid residues would be plausible, compatible with a role for TF participation in co-translational protein folding.     

Early encounters of a nascent membrane protein: specificity and timing of contacts inside and outside the ribosome

An unbiased photo-cross-linking approach was used to probe the "molecular path" of a growing nascent Escherichia coli inner membrane protein (IMP) from the peptidyl transferase center to the surface of the ribosome. The nascent chain was initially in proximity to the ribosomal proteins L4 and L22 and subsequently contacted L23, which is indicative of progression through the ribosome via the main ribosomal tunnel. The signal recognition particle (SRP) started to interact with the nascent IMP and to target the ribosome-nascent chain complex to the Sec-YidC complex in the inner membrane when maximally half of the transmembrane domain (TM) was exposed from the ribosomal exit. The combined data suggest a flexible tunnel that may accommodate partially folded nascent proteins and parts of the SRP and SecY. Intraribosomal contacts of the nascent chain were not influenced by the presence of a functional TM in the ribosome.     

Protein folding and translocation across the endoplasmic reticulum membrane

Proteins destined for secretion are translocated across or inserted into the endoplasmic reticulum membrane whereupon they fold and assemble to their native state before their subsequent transport to the Golgi apparatus. Proteins that fail to fold correctly are translocated back across the endoplasmic reticulum membrane to the cytosol where they become substrates for the cytosolic degradative machinery. Central to translocation is a protein pore in the membrane called the translocon that allows passage of proteins in and out of the endoplasmic reticulum. It is clear that the conformation of the polypeptide chain influences the translocation process and that there is a temporal relationship between modification of the chain, translocation and folding. This review will consider when and how the polypeptide chain folds, and how this might influence translocation into and out of the ER; and discuss how protein folding might affect post-translational modification of the polypeptide chain following translocation into the ER lumen.     

Protein folding and translocation across the endoplasmic reticulum membrane (Review)

Proteins destined for secretion are translocated across or inserted into the endoplasmic reticulum membrane whereupon they fold and assemble to their native state before their subsequent transport to the Golgi apparatus. Proteins that fail to fold correctly are translocated back across the endoplasmic reticulum membrane to the cytosol where they become substrates for the cytosolic degradative machinery. Central to translocation is a protein pore in the membrane called the translocon that allows passage of proteins in and out of the endoplasmic reticulum. It is clear that the conformation of the polypeptide chain influences the translocation process and that there is a temporal relationship between modification of the chain, translocation and folding. This review will consider when and how the polypeptide chain folds, and how this might influence translocation into and out of the ER; and discuss how protein folding might affect post-translational modification of the polypeptide chain following translocation into the ER lumen.     

Dissociation of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A action

Brefeldin A (BFA) has a profound effect on the structure of the Golgi apparatus, causing Golgi proteins to redistribute into the ER minutes after drug treatment. Here we describe the dissociation of a 110-kD cytoplasmically oriented peripheral membrane protein (Allan, V. J., and T. E. Kreis. 1986. J. Cell Biol. 103:2229-2239) from the Golgi apparatus as an early event in BFA action, preceding other morphologic changes. In contrast, other peripheral membrane proteins of the Golgi apparatus were not released but followed Golgi membrane into the ER during BFA treatment. The 110-kD protein remained widely dispersed throughout the cytoplasm during drug treatment, but upon removal of BFA it reassociated with membranes during reformation of the Golgi apparatus. Although a 30-s exposure to the drug was sufficient to cause the redistribution of the 110-kD protein, removal of the drug after this short exposure resulted in the reassociation of the 110-kD protein and no change in Golgi structure. If cells were exposed to BFA for 1 min or more, however, a portion of the Golgi membrane was committed to move into and out of the ER after removal of the drug. ATP depletion also caused the reversible release of the 110-kD protein, but without Golgi membrane redistribution into the ER. These findings suggest that the interaction between the 110-kD protein and the Golgi apparatus is dynamic and can be perturbed by metabolic changes or the drug BFA.     

Mechanistic Insight into the Reactivation of BCAII Enzyme from Denatured and Molten Globule States by Eukaryotic Ribosomes and Domain V rRNAs

In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events in vitro. Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process.