Plasma membrane targeting of chimeric intracisternal A-type particle polyproteins leads to particle release and specific activation of the viral proteinase.

Retrovirus morphogenesis involves assembly of structural Gag polyproteins with subsequent budding from the plasma membrane, followed by proteolytic cleavage by the viral proteinase (PR) and extracellular maturation to the infectious virion. Intracisternal A-type particles (IAPs) are defective retroviruses that assemble and bud at the membranes of the endoplasmic reticulum (ER), where they remain as immature particles consisting exclusively of uncleaved polyproteins. To analyze requirements for intracellular polyprotein transport and PR activation, we constructed deletion and substitution mutations in the IAP gag gene, including the putative ER-targeting signal. Mutant polyproteins were transported to various intracellular locations, including the nucleus, the cytoplasm, the ER, and the plasma membrane. Interestingly, assembly of capsid-like particle structures occurred at almost all sites. However, only those polyproteins transported to the plasma membrane were efficiently and specifically cleaved by viral PR, with cleavage occurring predominantly within the virus particle. Thus, at least in the experimental system presented here, retroviral particle assembly can occur at almost any location within the cell, while polyprotein processing and, consequently, virion maturation are confined to a specific cellular site. These results suggest that a factor restricted to the plasma membrane is required to trigger PR activation and maturation of infectious retroviruses.     

Human autoantibodies against the 54 kDa protein of the signal recognition particle block function at multiple stages

The 54 kDa subunit of the signal recognition particle (SRP54) binds to the signal sequences of nascent secretory and membrane proteins and it contributes to the targeting of these precursors to the membrane of the endoplasmic reticulum (ER). At the ER membrane, the binding of the signal recognition particle (SRP) to its receptor triggers the release of SRP54 from its bound signal sequence and the nascent polypeptide is transferred to the Sec61 translocon for insertion into, or translocation across, the ER membrane. In the current article, we have characterized the specificity of anti-SRP54 autoantibodies, which are highly characteristic of polymyositis patients, and investigated the effect of these autoantibodies on the SRP function in vitro. We found that the anti-SRP54 autoantibodies had a pronounced and specific inhibitory effect upon the translocation of the secretory protein preprolactin when analysed using a cell-free system. Our mapping studies showed that the anti-SRP54 autoantibodies bind to the amino-terminal SRP54 N-domain and to the central SRP54 G-domain, but do not bind to the carboxy-terminal M-domain that is known to bind ER signal sequences. Nevertheless, anti-SRP54 autoantibodies interfere with signal-sequence binding to SRP54, most probably by steric hindrance. When the effect of anti-SRP autoantibodies on protein targeting the ER membrane was further investigated, we found that the autoantibodies prevent the SRP receptor-mediated release of ER signal sequences from the SRP54 subunit. This observation supports a model where the binding of the homologous GTPase domains of SRP54 and the α-subunit of the SRP receptor to each other regulates the release of ER signal sequences from the SRP54 M-domain.     

Signal recognition particle mediates a transient elongation arrest of preprolactin in reticulocyte lysate

Signal recognition particle (SRP) is a ribonucleoprotein that functions in the targeting of ribosomes synthesizing presecretory proteins to the ER. SRP binds to the signal sequence as it emerges from the ribosome, and in wheat germ extracts, arrests further elongation. The translation arrest is released when SRP interacts with its receptor on the ER membrane. We show that the delay of elongation mediated by SRP is not unique to wheat germ translation extracts. Addition of mammalian SRP to reticulocyte lysates resulted in a delay of preprolactin synthesis due to increased ribosome pausing at specific sites on preprolactin mRNA. Addition of canine pancreatic microsomal membranes to reticulocyte lysates resulted in an acceleration of preprolactin synthesis, suggesting that the endogenous SRP present in the reticulocyte lysate also delays synthesis of secretory proteins.     

The affinity of signal recognition particle for presecretory proteins is dependent on nascent chain length.

We have developed an assay in which incomplete preprolactin chains of varying lengths are targeted to the endoplasmic reticulum (ER) membrane in an elongation independent manner. The reaction had the same molecular requirements as nascent chain translocation across the ER membrane, namely, it was signal recognition particle (SRP) dependent, and required the nascent chain to be present as peptidyl tRNA (i.e. most likely ribosome associated) and to have its signal sequence exposed outside the ribosome. We found that the efficiency of the targeting reaction dropped dramatically as the chains grew longer than 140 amino acids in length, which probably reflected a decrease in affinity of the nascent chain-ribosome complex for SRP. Thus at physiological SRP concentrations (10 nM) there appears a sharp cut-off point in the ability of these chains to be targeted, while at high SRP concentrations (270 nM) all chains could be targeted. In kinetic experiments, high concentrations of SRP were found to change the time in elongation after which translocation of the nascent polypeptide could no longer occur.     

Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking

We have studied the interaction between the signal sequence of nascent preprolactin and the signal recognition particle (SRP) during the initial events in protein translocation across the endoplasmic reticulum membrane. A new method of affinity labeling was used, whereby lysine residues, carrying the photoreactive group 4-(3-trifluoromethyldiazirino) benzoic acid in their side chains, are incorporated into a protein by means of modified lysyl-tRNA, and cross-linking to the interacting component is induced by irradiation. SRP interacts through its Mr 54,000 polypeptide component with the signal sequences of nascent preprolactin chains containing about 70 residues, and with decreasing affinity with longer chains as well; it causes inhibition of elongation. Binding of SRP is reversible and requires the nascent chain to be bound to a functional ribosome. SRP cross-linked to the signal sequence still inhibits elongation but does not prevent it completely. We conclude that SRP does not block the exit site of the polypeptide chain on the ribosome. The SRP receptor of the endoplasmic reticulum membrane displaces the signal sequence from SRP and, even if SRP is cross-linked, releases elongation arrest.     

Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes.     

GTPase domain of the 54-kD subunit of the mammalian signal recognition particle is required for protein translocation but not for signal sequence binding

The 54-kD subunit of the signal recognition particle (SRP54) binds to signal sequences of nascent secretory and transmembrane proteins. SRP54 consists of two separable domains, a 33-kD amino-terminal domain that contains a GTP-binding site (SRP54G) and a 22-kD carboxy-terminal domain (SRP54M) containing binding sites for both the signal sequence and SRP RNA. To examine the function of the two domains in more detail, we have purified SRP54M and used it to assemble a partial SRP that lacks the amino-terminal domain of SRP54 [SRP(-54G)]. This particle recognized signal sequences in two independent assays, albeit less efficiently than intact SRP. Analysis of the signal sequence binding activity of free SRP54 and SRP54M supports the conclusion that SRP54M binds signal sequences with lower affinity than the intact protein. In contrast, when SRP(-54G) was assayed for its ability to promote the translocation of preprolactin across microsomal membranes, it was completely inactive, apparently because it was unable to interact normally with the SRP receptor. These results imply that SRP54G plays an essential role in SRP-mediated targeting of nascent chain-ribosome complexes to the ER membrane and also influences signal sequence recognition, possibly by promoting a tighter association between signal sequences and SRP54M.     

Signal recognition particle mediates post-translational targeting in eukaryotes

Signal recognition particle (SRP) plays a central role in the delivery of classical secretory and membrane proteins to the endoplasmic reticulum (ER). All nascent chains studied to date dissociate from SRP once released from the ribosome, thereby supporting a strictly cotranslational mode of action for eukaryotic SRP. We now report a novel post-translational function for SRP in the targeting of tail-anchored (TA) proteins to the ER. TA proteins possess a hydrophobic membrane insertion sequence at their C-terminus such that it can only emerge from the ribosome after translation is terminated. We show that SRP can associate post-translationally with this type of ER-targeting signal, and deliver newly synthesised TA proteins to the ER membrane by a pathway dependent upon GTP and the SRP receptor. We find that dependency upon this SRP-dependent route is precursor specific, and propose a unifying model to describe the biogenesis of TA proteins in vivo.     

Evidence for a two-step mechanism involved in assembly of functional signal recognition particle receptor

The signal recognition particle (SRP) and SRP receptor act sequentially to target nascent secretory proteins to the membrane of the ER. The SRP receptor consists of two subunits, SR alpha and SR beta, both tightly associated with the ER membrane. To examine the biogenesis of the SRP receptor we have developed a cell-free assay system that reconstitutes SR alpha membrane assembly and permits both anchoring and functional properties to be assayed independently. Our experiments reveal a mechanism involving at least two distinct steps, targeting to the ER and anchoring of the targeted molecule on the cytoplasmic face of the membrane. Both steps can be reconstituted in vitro to restore translocation activity to ER microsomes inactivated by alkylation with N-ethyl-maleimide. The characteristics elucidated for this pathway distinguish it from SRP-dependent targeting of secretory proteins, SRP-independent ER translocation of proteins such as prepromellitin, and direct insertion mechanisms of the type exemplified by cytochrome b5.     

Inhibition of the biosynthesis of SRP polypeptides and secretory proteins by aflatoxin B1 can disrupt protein targeting

Cell culture and western blotting studies revealed that aflatoxin B(1) (AFB(1)) inhibits the biosynthesis of two of the constituent polypeptides of signal recognition particle (SRP) (SRP54 and 72). SRP escorts polyribosomes carrying signal peptides from free form in the cytosol to the bound form on endoplasmic reticulum (ER) membrane during protein targeting. These effects of AFB(1) on SRP biosynthesis may inhibit the formation of functional SRP. Our experiments have further shown that AFB(1) also inhibits the biosynthesis/translocation of a secretory protein, preprolactin, which fails to appear in the lumen of ER consequent to the treatment with this hepatocarcinogen. The results of the experiments presented in this article therefore enable us to infer for the first time that aflatoxin B(1) may inhibit the functioning of SRP as an escort and deplete the ER of polyribosomes for secretory protein synthesis. As these secretory proteins are important components of the plasma membrane, gap junctions and intercellular matrix, their absence from these locations could disturb cell to cell communication leading to tumorigenesis.     

Mutations in the N-terminal region of human immunodeficiency virus type 1 matrix protein block intracellular transport of the Gag precursor.

The matrix domain of human immunodeficiency virus type 1 Gag polyprotein was studied for its role in virus assembly. Deletion and substitution mutations caused a dramatic reduction in virus production. Mutant Gag polyproteins were myristoylated and had a high affinity for membrane association. Immunofluorescence staining revealed a large accumulation of mutant Gag precursors in the cytoplasm, while wild-type Gag proteins were primarily associated with the cell surface membrane. These results suggest a defect in intracellular transport of the mutant Gag precursors. Thus, in addition to myristoylation, the N-terminal region of the matrix domain is involved in determining Gag protein transport to the plasma membrane. Wild-type Gag polyproteins interacted with and efficiently packaged mutant Gag into virions. This finding is consistent with the hypothesis that intermolecular interaction of Gag polyproteins might occur in the cytoplasm prior to being transported to the assembly site on the plasma membrane.     

A two-step recognition of signal sequences determines the translocation efficiency of proteins.

The cytosolic and secreted, N-glycosylated, forms of plasminogen activator inhibitor-2 (PAI-2) are generated by facultative translocation. To study the molecular events that result in the bi-topological distribution of proteins, we determined in vitro the capacities of several signal sequences to bind the signal recognition particle (SRP) during targeting, and to promote vectorial transport of murine PAI-2 (mPAI-2). Interestingly, the six signal sequences we compared (mPAI-2 and three mutated derivatives thereof, ovalbumin and preprolactin) were found to have the differential activities in the two events. For example, the mPAI-2 signal sequence first binds SRP with moderate efficiency and secondly promotes the vectorial transport of only a fraction of the SRP-bound nascent chains. Our results provide evidence that the translocation efficiency of proteins can be controlled by the recognition of their signal sequences at two steps: during SRP-mediated targeting and during formation of a committed translocation complex. This second recognition may occur at several time points during the insertion/translocation step. In conclusion, signal sequences have a more complex structure than previously anticipated, allowing for multiple and independent interactions with the translocation machinery.     

The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane

The signal recognition particle receptor (SR) is required for the cotranslational targeting of both secretory and membrane proteins to the endoplasmic reticulum (ER) membrane. During targeting, the SR interacts with the signal recognition particle (SRP) which is bound to the signal sequence of the nascent protein chain. This interaction catalyzes the GTP-dependent transfer of the nascent chain from SRP to the protein translocation apparatus in the ER membrane. The SR is a heterodimeric protein comprised of a 69-kD subunit (SR alpha) and a 30- kD subunit (SR beta) which are associated with the ER membrane in an unknown manner. SR alpha and the 54-kD subunits of SRP (SRP54) each contain related GTPase domains which are required for SR and SRP function. Molecular cloning and sequencing of a cDNA encoding SR beta revealed that SR beta is a transmembrane protein and, like SR alpha and SRP54, is a member of the GTPase superfamily. Although SR beta defines its own GTPase subfamily, it is distantly related to ARF and Sar1. Using UV cross-linking, we confirm that SR beta binds GTP specifically. Proteolytic digestion experiments show that SR alpha is required for the interaction of SRP with SR. SR alpha appears to be peripherally associated with the ER membrane, and we suggest that SR beta, as an integral membrane protein, mediates the membrane association of SR alpha. The discovery of its guanine nucleotide-binding domain, however, makes it likely that its role is more complex than that of a passive anchor for SR alpha. These findings suggest that a cascade of three directly interacting GTPases functions during protein targeting to the ER membrane.     

Sec62 Protein Mediates Membrane Insertion and Orientation of Moderately Hydrophobic Signal Anchor Proteins in the Endoplasmic Reticulum (ER)

Nascent chains are known to be targeted to the endoplasmic reticulum membrane either by a signal recognition particle (SRP)-dependent co-translational or by an SRP-independent post-translational translocation route depending on signal sequences. Using a set of model and cellular proteins carrying an N-terminal signal anchor sequence of controlled hydrophobicity and yeast mutant strains defective in SRP or Sec62 function, the hydrophobicity-dependent targeting efficiency and targeting pathway preference were systematically evaluated. Our results suggest that an SRP-dependent co-translational and an SRP-independent post-translational translocation are not mutually exclusive for signal anchor proteins and that moderately hydrophobic ones require both SRP and Sec62 for proper targeting and translocation to the endoplasmic reticulum. Further, defect in Sec62 selectively reduced signal sequences inserted in an N_in-C_out (type II) membrane topology, implying an undiscovered role of Sec62 in regulating the orientation of the signal sequence in an early stage of translocation.     

Signal Recognition Particle and SecA Cooperate during Export of Secretory Proteins with Highly Hydrophobic Signal Sequences

The Sec translocon of bacterial plasma membranes mediates the linear translocation of secretory proteins as well as the lateral integration of membrane proteins. Integration of many membrane proteins occurs co-translationally via the signal recognition particle (SRP)-dependent targeting of ribosome-associated nascent chains to the Sec translocon. In contrast, translocation of classical secretory proteins across the Sec translocon is a post-translational event requiring no SRP but the motor protein SecA. Secretory proteins were, however, reported to utilize SRP in addition to SecA, if the hydrophobicity of their signal sequences exceeds a certain threshold value. Here we have analyzed transport of this subgroup of secretory proteins across the Sec translocon employing an entirely defined in vitro system. We thus found SecA to be both necessary and sufficient for translocation of secretory proteins with hydrophobic signal sequences, whereas SRP and its receptor improved translocation efficiency. This SRP-mediated boost of translocation is likely due to the early capture of the hydrophobic signal sequence by SRP as revealed by site-specific photo cross-linking of ribosome nascent chain complexes.     

Membrane protein topology of oleosin is constrained by its long hydrophobic domain.

Oleosin proteins from Arabidopsis assume a unique endoplasmic reticulum (ER) topology with a membrane-integrated hydrophobic (H) domain of 72 residues, flanked by two cytosolic hydrophilic domains. We have investigated the targeting and topological determinants present within the oleosin polypeptide sequence using ER-derived canine pancreatic microsomes. Our data indicate that oleosins are integrated into membranes by a cotranslational, translocon-mediated pathway. This is supported by the identification of two independent functional signal sequences in the H domain, and by demonstrating the involvement of the SRP receptor in membrane targeting. Oleosin topology was manipulated by the addition of an N-terminal cleavable signal sequence, resulting in translocation of the N terminus to the microsomal lumen. Surprisingly, the C terminus failed to translocate. Inhibition of C-terminal translocation was not dependent on either the sequence of hydrophobic segments in the H domain, the central proline knot motif or charges flanking the H domain. Therefore, the topological constraint results from the length and/or the hydrophobicity of the H domain, implying a general case that long hydrophobic spans are unable to translocate their C terminus to the ER lumen.     

Inefficient targeting to the endoplasmic reticulum by the signal recognition particle elicits selective defects in post-ER membrane trafficking

The signal recognition particle (SRP) is required for protein translocation into the endoplasmic reticulum (ER). With RNA interference we reduced its level about ten-fold in mammalian cells to study its cellular functions. Such low levels proved insufficient for efficient ER-targeting, since the accumulation of several proteins in the secretory pathway was specifically diminished. Although the cells looked unaffected, they displayed noticeable and selective defects in post-ER membrane trafficking. Specifically, the anterograde transport of VSV-G and the retrograde transport of the Shiga toxin B-subunit were stalled at the level of the Golgi whereas the endocytosed transferrin receptor failed to recycle to the plasma membrane. Endocytic membrane trafficking from the plasma membrane to lysosomes or Golgi was undisturbed and major morphological changes in the ER and the Golgi were undetectable at low resolution. Selective membrane trafficking defects were specifically suppressed under conditions when low levels of SRP became sufficient for efficient ER-targeting and are therefore a direct consequence of the lower targeting capacity of cells with reduced SRP levels. Selective post-ER membrane trafficking defects occur at SRP levels sufficient for survival suggesting that changes in SRP levels and their effects on post-ER membrane trafficking might serve as a mechanism to alter temporarily the localization of selected proteins.     

SecA is not required for signal recognition particle-mediated targeting and initial membrane insertion of a nascent inner membrane protein.

In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described. In vitro cross-linking studies have demonstrated that short nascent chains exposing a highly hydrophobic targeting signal interact with the SRP. This SRP, assisted by its receptor, FtsY, mediates the transfer to a common translocation site in the inner membrane that contains SecA, SecG, and SecY. Here we describe a further in vitro reconstitution of SRP-mediated membrane insertion in which purified ribosome-nascent chain-SRP complexes are targeted to the purified SecYEG complex contained in proteoliposomes in a process that requires the SRP-receptor FtsY and GTP. We found that in this system SecA and ATP are dispensable for both the transfer of the nascent inner membrane protein FtsQ to SecY and its stable membrane insertion. Release of the SRP from nascent FtsQ also occurred in the absence of SecYEG complex indicating a functional interaction of FtsY with lipids. These data suggest that SRP/FtsY and SecB/SecA constitute distinct targeting routes.     

The methionine-rich domain of the 54 kDa subunit of signal recognition particle is sufficient for the interaction with signal sequences.

The signal recognition particle (SRP) binds to signal sequences when they emerge from a translating ribosome and targets the complex of ribosome, nascent chain and SRP to the membrane of the rough endoplasmic reticulum (rER) allowing the co-translational translocation of the nascent chain. By photo-crosslinking it has been shown that the signal sequence of preprolactin (PPL) only interacts with the methionine-rich (M) domain of the 54 kDa protein subunit (SRP54) of SRP. Here we show that (i) a signal-anchor sequence is likewise crosslinked only to the methionine-rich domain of SRP54, (ii) free SRP54 can interact with signal sequences independently of the other components of SRP, (iii) its M domain suffices to perform this function, and (iv) an essentially intact M domain is required for signal sequence recognition. Alkylation of the N+G domain in intact SRP54 with N-ethyl maleimide (NEM), but not after cleavage with V8 protease, prevents the binding of a signal sequence to the M domain. This suggests a proximity between the N+G and M domains of SRP54 and raises the possibility that the role of the N+G domain may be to regulate the binding and/or the release of signal sequences.     

Regulation of ribosome detachment from the mammalian endoplasmic reticulum membrane

In current models, protein translocation in the endoplasmic reticulum (ER) occurs in the context of two cycles, the signal recognition particle (SRP) cycle and the ribosome cycle. Both SRP and ribosomes bind to the ER membrane as a consequence of the targeting process of translocation. Whereas SRP release from the ER membrane is regulated by the GTPase activities of SRP and the SRP receptor, ribosome release from the ER membrane is thought to occur in response to the termination of protein synthesis. We report that ER-bound ribosomes remain membrane-bound following the termination of protein synthesis and in the bound state can initiate the translation of secretory and cytoplasmic proteins. Two principal observations are reported. 1) Membrane-bound ribosomes engaged in the synthesis of proteins lacking a signal sequence are released from the ER membrane as ribosome-nascent polypeptide complexes. 2) Membrane-bound ribosomes translating secretory proteins can access the translocon in an SRP receptor-independent manner. We propose that ribosome release from the ER membrane occurs in the context of protein translation, with release occurring by default in the absence of productive nascent polypeptide-membrane interactions.