Translocation Of Secretory Proteins Research Articles

Secretory protein translocation in a neurospora crassa in vitro system. Hydrolysis of a nucleoside triphosphate is required for posttranslational translocation.

An in vitro translocation system has been reconstituted with subcellular fractions from the cell wall-less mutant of Neurospora crassa (fz;sg;os-1). Prepro alpha factor and invertase, secretory proteins from yeast, were faithfully translocated and glycosylated by Neurospora microsomes when presence cotranslationally in the Neurospora translation system. When presence cotranslationally in the Neurospora translation system, microsomes from canine pancreas(cRM) could also translocate and glycosylate the secretory proteins. However, salt-extracted cRM, which is depleted of canine signal recognition particle, could not. Furthermore, prepro alpha factor and a truncated form of invertase, containing the first 262-amino acid residues of the secretory invertase, were glycosylated by Neurospora microsomes posttranslationally, whereas only the truncated form of invertase was glycosylated by cRM when added posttranslationally. The full length invertase was not glycosylated posttranslationally. Posttranslational glycosylation of prepro alpha factor and of the truncated form of invertase is dependent on the hydrolysis of a nucleoside triphosphate. These data suggest that posttranslational glycosylation of prepro alpha factor occurs via a novel type of recognition mechanism which is either absent or ineffective in cRM.

GTP-dependent ADP-ribosylation of a 22 kDa protein in the endoplasmic reticulum membrane

Treatment of salt-stripped rough microsomal membranes from pancreas or liver with NAD and cholera toxin in the presence of GTP yields an ADP-ribosylated non-ribosomal 22 kDa protein. Membranes containing the modified protein are less active in the co-translational processing of secretory preproteins translated from isolated mRNA in a reticulocyte translation system, but signal peptidase activity is unchanged, suggesting that the 22 kDa protein is involved in the targetting or translocation of secretory proteins at the membrane of the endoplasmic reticulum.

Soluble factors stimulating secretory protein translocation in bacteria and yeast can substitute for each other.

mRNA for prepro-alpha-factor (pp alpha), a yeast secretory glycoprotein, was translated in a wheat germ cell-free system that was posttranslationally supplemented either with inverted vesicles from the plasma membrane of Escherichia coli (INV) or with microsomes from Saccharomyces cerevisiae. A postribosomal supernatant (PRS) from E. coli was found to stimulate translocation of pp alpha across the INV membrane. A yeast PRS could substitute for its E. coli counterpart. Likewise, an E. coli PRS could substitute for a yeast PRS and stimulate translocation of pp alpha across yeast microsomal membranes.

Nascent secretory polypeptides synthesized on Escherichia coli ribosomes are not translocated across mammalian endoplasmic reticulum.

Cell-free protein-synthesizing systems from Escherichia coli and wheat germ were compared for their capacity to support the translocation of secretory proteins across microsomal membranes derived from mammalian endoplasmic reticulum. Three different secretory proteins, two of bacterial and one of eucaryotic origin, were tested in this respect. In all three cases a contrast between the results in the eucaryotic and procaryotic protein-synthesizing systems was revealed. Whereas the eucaryotic system, as expected, supported the translocation of nascent secretory proteins across the microsomal membranes, the procaryotic system failed to do so. This failure was not due to the absence of a translocation-promoting activity or the presence of a translocation-blocking activity in the procaryotic system. These results demonstrate a specificity in the requirement of components of the protein-synthesizing machinery for protein translocation. These components might participate in forming a functional ribosome-membrane junction during protein translocation. The nascent secretory chain alone is not sufficient for making this junction, which might involve the postulated binding of the ribosome to the signal recognition particle or another component of the membrane.

Identification of signal sequence binding proteins integrated into the rough endoplasmic reticulum membrane.

An azidophenacyl derivative of a chemically synthesized consensus signal peptide has been prepared. The peptide, when photoactivated in the presence of rough or high-salt-stripped microsomes from pancreas, leads to inhibition of their activity in cotranslational processing of secretory pre-proteins translated from their mRNA in vitro. The peptide binds specifically with high affinity to components in the microsomal membranes from pancreas and liver, and photoreaction of a radioactive form of the azidophenacyl derivative leads to covalent linkage to yield two closely related radiolabelled proteins of Mr about 45,000. These proteins are integrated into the membrane, with large 30,000-Mr domains embedded into the phospholipid bilayer to which the signal peptide binds. A smaller, endopeptidase-sensitive, domain is exposed on the cytoplasmic surface of the microsomal vesicles. The specificity and selectivity of the binding of azidophenacyl-derivatized consensus signal peptide was demonstrated by concentration-dependent inhibition of photolabelling by the 'cold' synthetic consensus signal peptide and by a natural internal signal sequence cleaved and isolated from ovalbumin. The properties of the labelled 45,000-Mr protein-signal peptide complexes, i.e. mass, pI, ease of dissociation from the membrane by detergent or salts and immunological properties, distinguish them from other proteins, e.g. subunits of signal recognition particle, docking protein and signal peptidase, already known to be involved in targetting and processing of nascent secretory proteins at the rough endoplasmic reticulum membrane. Although the 45,000-Mr signal peptide binding protein displays properties similar to those of the signal peptidase, a component of the endoplasmic reticulum, the azido-derivatized consensus signal peptide does not interact with it. It is proposed that the endoplasmic reticulum proteins with which the azidophenacyl-derivatized consensus signal peptide interacts to yield the 45,000-Mr adducts may act as receptors for signals in nascent secretory pre-proteins in transduction of changes in the endoplasmic reticulum which bring about translocation of secretory protein across the membrane.

How proteins move across the endoplasmic reticulum membrane

How proteins move across the endoplasmic reticulum membrane

Characterization of secretory protein translocation: ribosome-membrane interaction in endoplasmic reticulum.

Secretory proteins are synthesized on ribosomes bound to the membrane of the endoplasmic reticulum (ER). After the selection of polysomes synthesizing secretory proteins and their direction to the membrane of the ER via signal recognition particle (SRP) and docking protein respectively, the polysomes become bound to the ER membrane via an unknown, protein-mediated mechanism. To identify proteins involved in protein translocation, beyond the (SRP-docking protein-mediated) recognition step, controlled proteolysis was used to functionally inactivate rough microsomes that had previously been depleted of docking protein. As the membranes were treated with increasing levels of protease, they lost their ability to be functionally reconstituted with the active cytoplasmic fragment of docking protein (DPf). This functional inactivation did not correlate with a loss of either signal peptidase activity, nor with the ability of the DPf to reassociate with the membrane. It did correlate, however, with a loss of the ability of the microsomes to bind ribosomes. Ribophorins are putative ribosome-binding proteins. Immunoblots developed with monoclonal antibodies against canine ribophorins I and II demonstrated that no correlation exists between the protease-induced inability to bind ribosomes and the integrity of the ribophorins. Ribophorin I was 85% resistant and ribophorin II 100% resistant to the levels of protease needed to totally eliminate ribosome binding. Moreover, no direct association was found between ribophorins and ribosomes; upon detergent solubilization at low salt concentrations, ribophorins could be sedimented in the presence or absence of ribosomes. Finally, the alkylating agent N-ethylmaleimide was shown to be capable of inhibiting translocation (beyond the SRP-docking protein-mediated recognition step), but had no affect on the ability of ribosomes to bind to ER membranes. We conclude that potentially two additional proteinaceous components, as yet unidentified, are involved in protein translocation. One is protease sensitive and possibly involved in ribosome binding, the other is N-ethylmaleimide sensitive and of unknown function.

Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis.

We describe an in vitro system with all components derived from the yeast Saccharomyces cerevisiae that can translocate a yeast secretory protein across microsomal membranes. In vitro transcribed prepro-alpha-factor mRNA served to program a membrane-depleted yeast translation system. Translocation and core glycosylation of prepro-alpha-factor were observed when yeast microsomal membranes were added during or after translation. A membrane potential is not required for translocation. However, ATP is required for translocation and nonhydrolyzable analogues of ATP cannot serve as a substitute. These findings suggest that ATP hydrolysis may supply the energy required for translocation of proteins across the endoplasmic reticulum.

Translocation of nascent secretory proteins across membranes can occur late in translation.

Signal recognition particle (SRP) causes an arrest in the translation of nascent secretory proteins in a wheat germ cell-free system. In order to examine at what point during the synthesis of a secretory protein its translocation across the endoplasmic reticulum (ER) membrane can occur, SRP was used to arrest nascent chain elongation at various times during a synchronous translation, thus allowing the generation of nascent chains of increasing length. It was found that SRP can still bring about an arrest as late as when an average of two-thirds of nascent IgG light chain was completed. Rough microsomes were added to translations blocked with SRP to determine if such relatively long nascent chains could still be translocated across the membrane. It was found that nascent chains which had been arrested by SRP, regardless of their length, could be translocated into rough microsomes. In the case of IgG light chain, translocation levels of 50% were still observed with nascent chains corresponding to as much as 70-75% of the intact preprotein. Similar results were observed for the nascent bovine prolactin precursor. These results demonstrate that the synthesis of secretory proteins can be uncoupled from their translocation, and that fairly large nascent chains are capable of crossing the membrane of the ER post-translationally.

The signal sequence of nascent preprolactin interacts with the 54K polypeptide of the signal recognition particle.

Hydrophobic signal sequences direct the translocation of nascent secretory proteins and many membrane proteins across the membrane of the endoplasmic reticulum. Initiation of this process involves the signal recognition particle (SRP), which consists of six polypeptide chains and a 7S RNA and interacts with ribosomes carrying nascent secretory polypeptide chains. In the case of aminoterminal, cleavable signal sequences, in the absence of microsomal membranes it exerts a site-specific translational arrest in vitro. The size of the arrested fragment (60-70 amino-acid residues) suggests that elongation stops when the signal sequence has emerged fully from the ribosome. However, a direct interaction between the signal sequence and SRP has not previously been demonstrated and has even been questioned recently. We now show for the first time a direct interaction between the signal sequence of a secretory protein and a component of SRP, the 45K polypeptide (relative molecular mass (Mr) 54,000). This was achieved by means of a new method of affinity labelling which involves the translational incorporation of an amino acid, carrying a photoreactive group, into nascent polypeptides.

Removal of the Alu structural domain from signal recognition particle leaves its protein translocation activity intact

Alu-like elements comprise the most abundant family of interspersed repetitive sequences in primates and rodents, and contain many features of processed genes, suggesting that they were initially derived by reverse transcription of processed RNA transcripts. Transcripts containing Alu family members are represented in heterologous nuclear RNAs, cytoplasmic messenger RNAs and small RNAs, although nothing is known about their function. Evolutionary studies strongly suggest that the parent RNA for the Alu-like elements is the highly conserved 7SL RNA, which is an essential component of signal recognition particle (SRP), a small cytoplasmic ribonucleoprotein whose function is the targeting of nascent secretory and membrane proteins to the rough endoplasmic reticulum (for a review see ref. 6). 7SL RNA is composed of both unique and Alu-like sequences. SRP is rod-shaped and, in addition to its RNA, contains four proteins (two monomers composed of a polypeptide of relative molecular mass (Mr) 19,000 (19K) and one of 54K, and two heterodimers, one composed of a 9K and a 14K polypeptide, and the other composed of a 68K and a 72K polypeptide, respectively). The RNA moiety is required for SRP activity, as well as for structural integrity of the particle. To investigate whether the Alu-like segments of 7SL RNA have a specific role in SRP activity, we have now purified and analysed a SRP subparticle that is created upon extensive digestion with micrococcal nuclease and entirely lacks the Alu-like sequences. We find that it contains the 72/68K, 54K and 19K proteins tightly bound, but lacks the 9/14K protein. In vitro activity assays demonstrated that the subparticle could still promote secretory protein translocation across the microsomal membrane, but could no longer trigger an arrest of pre-secretory protein synthesis. Re-addition of the 9/14K protein did not restore the elongation arrest. We conclude that the region of SRP comprised of the Alu-like RNA and the 9/14K protein exists in a distinct structural domain which is not required for the protein translocation promoted by SRP but apparently confers elongation-arresting activity on the particle.

Transfer of Secretory Proteins through the Membrane of the Endoplasmic Reticulum

Publisher Summary Secretory proteins are synthesized on ribosomes located in the cytoplasm and are released from the cell as a fairly homogeneous, highly concentrated population of molecules. Secretory proteins can be localized in the lumen of the endoplasmic reticulum (ER) very early in their existence. Translocation of secretory proteins from the cytoplasmic to the luminal side of the rough ER membrane is an essential feature of the transport of these molecules. The information for the association of the mRNAs encoding secretory proteins to the membrane is contained within secretory protein mRNAs itself. mRNA encoding a secretory protein contains the signal for its ultimate association with the membrane. All secretory protein mRNAs encode a transient N-terminal peptide. This short amino acid sequence provides a signal, which results in the formation of membrane-bound polysomes and, ultimately, the translocation across the membrane. This chapter presents the results of studies, which deal with various individual aspects of protein translocation. It also describes signal sequences, signal recognition particle, docking protein, ribosome binding, signal peptidase, and the cotranslational covalent modifications, which are carried out in the rough ER.

Translocation of secretory proteins across the microsomal membrane occurs through an environment accessible to aqueous perturbants

We have characterized the association of a nascent secretory protein with the microsomal membrane at two distinct stages in cell-free synthesis and translocation. Stage one corresponded to a nascent chain of ∼70 residues generated via elongation arrest by the signal recognition particle (SRP). Binding to microsomal membranes occurred independently of chain elongation and required SRP receptor. Following binding, the 70-mer remained attached to the membrane after extraction of the ribosome. However, protein denaturants (4 M urea or alkaline pH) extracted the 70-mer from the membrane. Stage two of synthesis corresponded to nascent chains of ∼158 residues generated by oligonucleotide-mediated hybrid arrest of translation. Again, these partially translocated nascent chains were extracted by 4 M urea. Therefore, the initial interaction of the signal sequence with the membrane as well as subsequent chain conductance occur in a microenvironment that is accessible to aqueous reagents. Thus, both processes probably require integral membrane proteins.

A structural and functional analysis of the docking protein. Characterization of active domains by proteolysis and specific antibodies.

Docking protein is a 73-kDa integral membrane protein of the rough endoplasmic reticulum. It is essential for translocation of nascent secretory proteins into the lumen of the endoplasmic reticulum. Monoclonal and polyclonal antibodies have been generated which, in conjunction with limited proteolysis, have been used to characterize several subspecies of docking protein. These proteolytic fragments have been analyzed with respect to the various functions ascribed to docking protein which can be assayed in vitro. Proteolytic digestion of membrane-associated or of affinity-purified intact docking protein showed that: elastase cleavage generates a 59-kDa soluble fragment and one of 14 kDa which contains the membrane anchoring domain; trypsin as well as endogenous proteolysis generates a 46-kDa fragment, leaving a 27-kDa domain containing the membrane anchor. This 27-kDa fragment can be reduced to a 13- and a 14-kDa piece by elastase digestion. The characteristics of these various subspecies were examined. The 59-kDa soluble fragment, which can reconstitute full translocation activity to docking protein-depleted microsomes (Meyer, D. I., and Dobberstein, B. (1980) J. Cell Biol. 87, 503-508) was capable of releasing a signal recognition particle-mediated translation arrest. The 46-kDa fragment was neither able to reassociate with nor to reconstitute the activity of docking protein-depleted microsomes. Moreover this fragment was unable to release a signal recognition particle-mediated arrest. This suggests that the 13-kDa fragment (the difference between 46 and 59 kDa) is both essential for association with the membrane, and for the release of translation arrests.

Elongation arrest is not a prerequisite for secretory protein translocation across the microsomal membrane.

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL RNA. It was previously shown to promote the co-translational translocation of secretory proteins across the endoplasmic reticulum by (a) arresting the elongation of the presecretory nascent chain at a specific point, and (b) interacting with the SRP receptor, an integral membrane protein of the endoplasmic reticulum which is active in releasing the elongation arrest. Recently a procedure was designed by which the particle could be disassembled into its protein and RNA components. We have further separated the SRP proteins into four homogeneous fractions. When recombined with each other and with 7SL RNA, they formed fully active SRP. Particles missing specific proteins were assembled in the hope that some of these would retain some functional activity. SRP(-9/14), the particle lacking the 9-kD and 14-kD polypeptides, was fully active in promoting translocation, but was completely inactive in elongation arrest. This implied that elongation arrest is not a prerequisite for protein translocation. SRP receptor was required for SRP(-9/14)-mediated translocation to occur, and thus must play some role in the translocation process in addition to releasing the elongation arrest.

Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. I. Functional tests on rat liver microsomal subfractions.

A preparation of rat liver microsomes containing 70% of the total cellular endoplasmic reticulum (ER) membranes was subfractionated by isopycnic density centrifugation. Twelve subfractions of different ribosome content ranging in density from 1.06 to 1.29 were obtained and analyzed with respect to marker enzymes, RNA, and protein content, as well as the capacity of these membranes to bind 80S ribosomes in vitro. After removal of native polysomes from these microsomal subfractions by puromycin in a buffer of high ionic strength their capacity to rebind 80S ribosomes approached levels found in the corresponding native membranes before ribosome stripping. This indicates that in vitro rebinding of ribosomes occurs to the same sites occupied in the cell by membrane-bound polysomes. Microsomes in the microsomal subfractions were also tested for their capacity to effect the translocation of nascent secretory proteins into the microsomal lumen utilizing a rabbit reticulocyte translation system programmed with mRNA coding for the precursor of human placental lactogen. Membranes from microsomes with the higher isopycnic density and a high ribosome content showed the highest translocation activity, whereas membranes derived from smooth microsomes had only a very low translocation activity. These results indicate the membranes of the rough and smooth portions of the endoplasmic reticulum are functionally differentiated so that sites for ribosome binding and the translocation of nascent polypeptides are segregated to the rough domain of the organelle.

Secretion of a wheat α-amylase expressed in yeast

The translocation of secretory proteins across the endoplasmic reticulum involves the recognition and cleavage of an amino-terminal extension called the signal sequence1. The structure of signal peptides appears to be ubiquitous in having a very hydrophobic central core2, so that the signal sequence in secretory proteins from one organism could possibly be recognized by the processing and transport apparatus of another. We therefore wished to investigate whether a protein, α-amylase, one of several hydrolytic enzymes secreted from the aleurone of wheat into the endosperm during germination, could be processed and secreted in an active form from the yeast Saccharomyces cerevisiae, secretion being dependent upon the plant signal sequence. Here, synthesis of α-amylase was by inserting a cDNA clone coding for the entire α-amylase structural gene3 into a yeast expression vector4. The α-amylase protein coded for by this gene fusion has the signal sequence located internally, not at the N-terminal end of the polypeptide. Nevertheless, it is processed and the processed form is secreted into the medium in an active form. There are potential industrial applications for yeast that secrete a functional α-amylase.

Transient involvement of signal recognition particle and its receptor in the microsomal membrane prior to protein translocation

We have shown that neither the signal recognition particle (SRP) nor the SRP receptor is directly involved in the maintenance of a ribosome-membrane junction for the translocation of secretory proteins. We found that the purified SRP receptor releases the signal-sequence-induced and SRP-mediated elongation arrest of synthesis by displacing SRP from the ribosome. This SRP displacement was not accompanied by binding of the SRP receptor to the ribosome. Using intact microsomal membranes as a source of the SRP receptor, we found that both SRP displacement and binding of the elongation-arrested ribosome to the membrane can occur at 0°C, by a mechanism that is independent of chain elongation.

Translocation of a lysosomal enzyme across the microsomal membrane requires signal recognition particle

Signal recognition particle (SRP), an 11S ribonucleoprotein (Walter and Blobel (1982) Nature 299, 691–698), is required for translocation of secretory proteins across microsomal membranes (Walter and Blobel (1980) Proc. Natl. Acad. Sci. USA 77, 7112–7116) and for asymmetric integration into microsomal membranes of a transmembrane protein (Anderson et al., (1982) J. Cell Biol. 93, 501–506). We demonstrate here that SRP is also required for translocation of the lysosomal protease cathepsin D across microsomal membranes.

54] Proteins mediating vectorial translocation: Purification of the active domain of the endoplasmic reticulum docking protein

This chapter discusses and presents the assay system and the procedures needed to isolate and purify the active domain of the docking protein from isolated rough microsomes. The processing of nascent secretory proteins involves cotranslational cleavage of the signal sequence on the luminal side of microsomal vesicles. The general principle of using dog pancreas microsomes to reconstitute translocation of secretory proteins must be slightly modified to allow the addition of isolated factors to the system and the membranes. Purification of docking protein involves preparation of stripped rough microsomes, detergent extraction of stripped rough microsomes (RM), elastase extraction of triton-insoluble membrane material, purification of the fragment of docking protein (DP f ) from elastase extract (EE) using ion-exchange chromatography, and gel filtration of CM-bound EE. The protein composition of fractions is best determined by polyacrylamide gel electrophoresis and staining with either Coomassie Blue or silver.