Integration of membrane proteins into the endoplasmic reticulum requires GTP

We have examined the requirement for ribonucleotides and ribonucleotide triphosphate hydrolysis during early events in the membrane integration of two membrane proteins: the G protein of vesicular stomatitis virus and the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus. Both proteins contain a single transmembrane-spanning segment but are integrated in the membrane with opposite orientations. The G protein has an amino-terminal signal sequence and a stop-transfer sequence located near the carboxy terminus. The HN glycoprotein has a single sequence near the amino terminus that functions as both a signal-sequence and a transmembrane-spanning segment. Membrane insertion was explored using a cell-free system directed by transcribed mRNAs encoding amino-terminal segments of the two proteins. Ribosome-bound nascent polypeptides were assembled, ribonucleotides were removed by gel filtration chromatography, and the ribosomes were incubated with microsomal membranes under conditions of defined ribonucleotide content. Nascent chain insertion into the membrane required the presence of both the signal recognition particle and a functional signal recognition particle receptor. In the absence of ribonucleotides, insertion of nascent membrane proteins was not detected. GTP or nonhydrolyzable GTP analogues promoted efficient insertion, while ATP was comparatively ineffective. Surprisingly, the majority of the HN nascent chain remained ribosome associated after puromycin treatment. Ribosome-associated HN nascent chains remained competent for membrane insertion, while free HN chains were not competent. We conclude that a GTP binding protein performs an essential function during ribosome-dependent insertion of membrane proteins into the endoplasmic reticulum that is unrelated to protein synthesis.     

Mutants of the membrane-binding region of Semliki Forest virus E2 protein. II. Topology and membrane binding

The p62/E2 protein of Semliki Forest virus (SFV) is a typical transmembrane glycoprotein, with an amino-terminal lumenal domain, a transmembrane (hydrophobic) domain, and a carboxy-terminal cytoplasmic domain (or tail). Our hypothesis has been that the membrane-binding polypeptide region (membrane anchor) of this protein consists of both the transmembrane domain and the adjacent positively charged peptide, Arg-Ser-Lys, which is part of the cytoplasmic domain. We have investigated three anchor mutants of the p62 protein with respect to both their disposition and their stability in cell membranes. The construction of the three mutants has been described (Cutler, D.F., and H. Garoff, J. Cell Biol., 102:889-901). They are as follows: A1, changing the basic charge cluster from Arg-Ser-Lys(+2) to Gly-Ser-Glu(-1); A2, replacing an Ala in the middle of the hydrophobic stretch with a Glu; A3, changing the charge cluster from Arg-Ser-Lys(+2) to Gly-Ser-Met(0). All three mutants retain the transmembrane configuration of the wild-type p62. In a cell homogenate they have a cytoplasmic domain that is accessible to protease. In living cells an anti-peptide antibody specific for the cytoplasmic tail of p62 reacts with the tails of both wild-type and mutant p62s following its introduction into the cytoplasm. All three mutant proteins have Triton X-114 binding properties similar to the wild-type p62. However, when the membranes of cells expressing the three mutants or the wild-type p62 protein are washed with sodium carbonate, pH 11.5, three to four times as much mutant protein as wild-type p62 is released from the membranes. Thus the stability in cell membranes of the three mutant p62 proteins is significantly reduced.     

Requirements for the membrane insertion of signal-anchor type proteins

Proteins which are inserted and anchored in the membrane of the ER by an uncleaved signal-anchor sequence can assume two final orientations. Type I signal-anchor proteins translocate the NH2 terminus across the membrane while type II signal-anchor proteins translocate the COOH terminus. We investigated the requirements for cytosolic protein components and nucleotides for the membrane targeting and insertion of single-spanning type I signal-anchor proteins. Besides the ribosome, signal recognition particle (SRP), GTP, and rough microsomes (RMs) no other components were found to be required. The GTP analogue GMPPNP could substitute for GTP in supporting the membrane insertion of IMC-CAT. By using a photocrosslinking assay we show that for secreted, type I and type II signal-anchor proteins the presence of both GTP and RMs is required for the release of the nascent chain from the 54-kD subunit of SRP. For two of the proteins studied the release of the nascent chain from SRP54 was accompanied by a new interaction with components of the ER. We conclude that the GTP-dependent release of the nascent chain from SRP54 occurs in an identical manner for each of the proteins studied.     

Intracellular processing and transport of NH2-terminally truncated forms of a hemagglutinin-neuraminidase type II glycoprotein

Six amino-terminal deletion mutants of the NH2-terminally anchored (type II orientation) hemagglutinin-neuraminidase (HN) protein of parainfluenza virus type 3 were expressed in tissue culture by recombinant SV-40 viruses. The mutations consisted of progressive deletions of the cytoplasmic domain and, in some cases, of the hydrophobic signal/anchor. Three activities were dissociated for the signal/anchor: membrane insertion, translocation, and anchoring/transport. HN protein lacking the entire cytoplasmic tail was inserted efficiently into the membrane of the endoplasmic reticulum but was translocated inefficiently into the lumen. However, the small amounts that were successfully translocated appeared to be processed subsequently in a manner indistinguishable from that of parental HN. Thus, the cytoplasmic domain was not required for maturation of this type II glycoprotein. Progressive deletions into the membrane anchor restored efficient translocation, indicating that the NH2-terminal 44 amino acids were fully dispensable for membrane insertion and translocation and that a 10-amino acid hydrophobic signal sequence was sufficient for both activities. These latter HN molecules appeared to be folded authentically as assayed by hemagglutination activity, reactivity with a conformation-specific antiserum, correct formation of intramolecular disulfide bonds, and homooligomerization. However, most (85-90%) of these molecules accumulated in the ER. This showed that folding and oligomerization into a biologically active form, which presumably represents a virion spike, occurs essentially to completion within that compartment but is not sufficient for efficient transport through the exocytotic pathway. Protein transport also appeared to depend on the structure of the membrane anchor. These latter mutants were not stably integrated in the membrane, and the small proportion (10-15%) that was processed through the exocytotic pathway was secreted. The maturation steps and some of the effects of mutations described here for a type II glycoprotein resemble previous observations for prototypic type I glycoproteins and are indicative of close similarities in these processes for proteins of both membrane orientations.     

Deletions in hydrophilic domains of subunit a from the Escherichia coli F1F0-ATP synthase interfere with membrane insertion or F0 assembly.

The a subunit is a membrane component of the F1F0-ATP synthase from Escherichia coli. Regions of a which appear important for membrane insertion or F0 assembly have been identified by analysis of both deletion mutants and fusion proteins which link the mutant a subunits to alkaline phosphatase. This analysis suggests the hydrophilic, amino-terminal domain of a is required for proper membrane targeting and/or insertion of the nascent polypeptide. In addition, the subcellular fractionation of four different a subunit-beta-galactosidase fusion proteins suggests this domain is localized to the periplasm, in agreement with a proposed topological model of the protein (Lewis, M.J., Chang, J.A., and Simoni, R.D. (1990) J. Biol. Chem. 265, 10541-10550). Deletions within the next three putative loops of a appear to have no significant effect on membrane targeting or insertion. Rather, they seem to interfere with the subsequent assembly of a functional enzyme.     

Discrete nascent chain lengths are required for the insertion of presecretory proteins into microsomal membranes

Ribosomes synthesizing nascent secretory proteins are targeted to the membrane by the signal recognition particle (SRP), a small ribonucleoprotein that binds to the signal peptide as it emerges from the ribosome. SRP arrests further elongation, causing ribosomes to stack behind the arrested ribosome. Upon interaction of SRP with its receptor on the ER membrane, the translation arrest is released and the ribosome becomes bound to the ER membrane. We have examined the distribution of unattached and membrane-bound ribosomes during the translation of mRNAs encoding two secretory proteins, bovine preprolactin and rat preproinsulin I. We find that the enhancement of ribosome stacking that occurs when SRP arrests translation of these proteins is relaxed in the presence of microsomal membranes. We also demonstrate that two previously described populations of membrane- associated ribosomes, distinguished by their sensitivity to high salt or EDTA extraction, correspond to ribosomes that have synthesized differing lengths of the nascent polypeptide. This analysis has revealed that nascent chain insertion into the membrane begins at distinct points for different presecretory proteins.     

Targeting and insertion of heterologous membrane proteins in E. coli

Membrane targeting and insertion of the archaeal Halobacter halobium proton pump bacterioopsin (Bop) and the human melanocortin 4 receptor (MC(4)R) were studied in vitro, using E. coli components for protein synthesis, membrane targeting and insertion. These heterologous proteins are targeted to E. coli membranes in a signal recognition particle (SRP) dependent manner and inserted into the membrane co-translationally. Furthermore, we demonstrate that nascent chains of Bop and MC(4)R first interact with SecY and then with YidC as they move through the translocon. Our results suggest that the initial stages of membrane targeting and insertion of heterologous proteins in E. coli proceed by the pathway used for native E. coli membrane proteins. No significant pausing of protein elongation was observed in the presence of E. coli SRP, in line with the suggestion that translational arrest requires an Alu domain, which is absent in SRP from E. coli.     

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.     

Membrane topology of the Bcl-2 proto-oncogenic protein demonstrated in vitro

The Bcl-2 oncogenic protein was synthesized in vitro and shown to post-translationally integrate asymmetrically into microsomal membranes with no requirement for an amino-terminal signal sequence. Instead, a carboxyl-terminal hydrophobic domain of Bcl-2 served as an insertion sequence essential for membrane assembly since a Bcl-2 mutant lacking this domain completely lost its ability to associate with microsomal membranes. The data demonstrate that Bcl-2 is tightly associated with the lipid bilayer with the nature of an integral membrane protein. The membrane orientation of Bcl-2 was determined using a protease protection assay, which showed that it is predominantly localized to the cytoplasmic face of membranes. A similar type of membrane processing has been shown for cytochrome b5 and also suggested for the viral oncogenic protein polyoma middle-T antigen.     

Requirements for the insertion of the Sindbis envelope glycoproteins into the endoplasmic reticulum membrane

Previous work has shown that the Sindbis structural proteins, core, the internal protein, and PE2 and E1, the integral membrane glycoproteins are synthesized as a polyprotein from a 26S mRNA; core PE2 and E1 are derived by proteolytic cleavage of a nascent chain. Newly synthesized core protein remains on the cytoplasmic side of the endoplasmic reticulum while newly synthesized PE2 and E1 are inserted into the lipid bilayer, presumably via their amino-termini. PE2 and E1 are glycosylated as nascent chains. Here, we examine a temperature-sensitive mutant of Sindbis virus which fails to cleave the structural proteins, resulting in the production of a polyprotein of 130,000 mol wt in which the amino-termini of PE2 and E1 are internal to the protein. Although the envelope sequences are present in this protein, it is not inserted into the endoplasmic reticulum bilayer, but remains on the cytoplasmic side as does the core protein in cells infected with wild-type Sindbis virus. We have also examined the fate of PE2 and E1 in cells treated with tunicamycin, an inhibitor of glycosylation. Unglycosylated PE2 and E1 are inserted normally into the lipid bilayer as are the glycosylated proteins. These results are consistent with the notion that a specific amino-terminal sequence is required for the proper insertion of membrane proteins into the endoplasmic reticulum bilayer, but that glycosylation is not required for this insertion.     

Transposition of domains between the M2 and HN viral membrane proteins results in polypeptides which can adopt more than one membrane orientation

The influenza A virus M2 polypeptide is a small integral membrane protein that does not contain a cleaved signal sequence, but is unusual in that it assumes the membrane orientation of a class I integral membrane protein with an NH2-terminal ectodomain and a COOH-terminal cytoplasmic tail. To determine the domains of M2 involved in specifying membrane orientation, hybrid genes were constructed and expressed in which regions of the M2 protein were linked to portions of the paramyxovirus HN and SH proteins, two class II integral membrane proteins that adopt the opposite orientation in membranes from M2. A hybrid protein (MgMH) consisting of the M2 NH2-terminal and membrane-spanning domains linked precisely to the HN COOH-terminal ectodomain was found in cells in two forms: integrated into membranes in the M2 topology or completely translocated across the endoplasmic reticulum membrane and ultimately secreted from the cell. The finding of a soluble form suggested that in this hybrid protein the anchor function of the M2 signal/anchor domain can be overridden. A second hybrid which contained the M2 NH2 terminus linked to the HN signal anchor and ectodomain (MgHH) was found in both the M2 and the HN orientation, suggesting that the M2 NH2 terminus was capable of reversing the topology of a class II membrane protein. The exchange of the M2 signal/anchor domain with that of SH resulted in a hybrid protein which assumed only the M2 topology. Thus, all these data suggest that the NH2-terminal 24 residues to M2 are important for directing the unusual membrane topology of the M2 protein. These data are discussed in relationship to the loop model for insertion of proteins into membranes and the role of charged residues as a factor in determining orientation.     

Evidence for mixed membrane topology of the newcastle disease virus fusion protein

The synthesis of the Newcastle disease virus (NDV) fusion (F) protein in a cell-free protein-synthesizing system containing membranes was characterized. The membrane-associated products were in at least two different topological forms with respect to the membranes. The properties of one form were consistent with the expected membrane insertion as a classical type 1 glycoprotein. This form of the protein was fully glycosylated, and sequences amino terminal to the transmembrane domain were protected from protease digestion by the membranes. The second form of membrane-associated F protein was partially glycosylated and partially protected from protease digestion by the membranes. Protease digestion resulted in a 23-kDa protease-protected polypeptide derived from F2 sequences and sequences from the amino-terminal end of the F1 domain. Furthermore, a 10-kDa polypeptide derived from the cytoplasmic domain (CT) was also protected from protease digestion by the membranes. Protease resistance of the 23- and 10-kDa polypeptides suggested that this second form of F protein inserted in membranes in a polytopic conformation with both the amino-terminal end and the carboxyl-terminal end translocated across membranes. To determine if this second form of the fusion protein could be found in cells expressing the F protein, two different approaches were taken. A polypeptide with the size of the partially translocated F protein was detected by Western analysis of proteins in total-cell extracts of NDV strain B1 (avirulent)-infected Cos-7 cells. Using antibodies raised against a peptide with sequences from the cytoplasmic domain, CT sequences were detected on surfaces of F protein-expressing Cos-7 cells by immunofluorescence and by flow cytometry. This antibody also inhibited the fusion of red blood cells to cells expressing F and HN proteins. These results suggest that NDV F protein made both in a cell-free system and in Cos-7 cells may exist in two topological forms with respect to membranes and that the second form of the protein may be involved in cell-cell fusion.     

Defective assembly and intracellular transport of mutant paramyxovirus hemagglutinin-neuraminidase proteins containing altered cytoplasmic domains.

The hemagglutinin-neuraminidase (HN) integral membrane protein of paramyxoviruses is expressed at the cell surface as a tetramer consisting of a pair of disulfide-linked dimers. HN has a large C-terminal ectodomain, a 19-residue uncleaved signal-anchor domain, and a 17-residue N-terminal cytoplasmic tail. Various mutant HN genes were constructed to examine the role of residues flanking the signal-anchor domain, including the cytoplasmic tail, on assembly and intracellular transport of the HN glycoprotein. Expression of the altered genes showed that by 90 min after synthesis the majority of the mutant HN proteins were in a conformationally mature form as assayed by their reactivity with conformation-specific monoclonal antibodies. However, the mutant proteins showed varied endoplasmic reticulum-to-Golgi apparatus transport rates, ranging from that of wild-type HN (t1/2 approximately 90 min) to slowly transported molecules (t1/2 approximately 5 h) and to molecules in which transport was not detected. Pulse-chase experiments indicated that the altered HN molecules had a specific and transient interaction with the resident endoplasmic reticulum protein GRP78-BiP, and thus the altered HN molecules were not retained in the endoplasmic reticulum by a prolonged interaction with GRP78-BiP. Sucrose density gradient sedimentation analysis of the mutant HN molecules indicated that they all had an oligomeric form that differed from that of wild-type HN; most of the molecules were found as disulfide-linked dimers rather than as tetramers. These data suggest that the HN cytoplasmic tail may function in the assembly of the final transport-competent oligomeric form of HN and that mutant HN molecules with seemingly properly folded ectodomains are retained in the endoplasmic reticulum by an as yet unidentified mechanism. The possible role of the HN cytoplasmic tail as a signal for intracellular transport is discussed.     

The Srp54 GTPase is essential for protein export in the fission yeast Schizosaccharomyces pombe.

Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein required for targeting a subset of presecretory proteins to the endoplasmic reticulum (ER) membrane. Here we report the results of a series of experiments to define the function of the Schizosaccharomyces pombe homolog of the 54-kDa subunit of mammalian SRP. One-step gene disruption reveals that the Srp54 protein, like SRP RNA, is essential for viability in S. pombe. Precursor to the secretory protein acid phosphatase accumulates in cells in which Srp54 synthesis has been repressed under the control of a regulated promoter, indicating that S. pombe SRP functions in protein targeting. In common with other Srp54 homologs, the S. pombe protein has a modular structure consisting of an amino-terminal G (GTPase) domain and a carboxyl-terminal M (methionine-rich) domain. We have analyzed the effects of 17 site-specific mutations designed to alter the function of each of the four GTPase consensus motifs individually. Several alleles, including some with relatively conservative amino acid substitutions, confer lethal or conditional phenotypes, indicating that GTP binding and hydrolysis are critical to the in vivo role of the protein. Two mutations (R to L at position 194 [R194L] and R194H) which were designed, by analogy to oncogenic mutations in rats, to dramatically decrease the catalytic rate and one (T248N) predicted to alter nucleotide binding specificity produce proteins that are unable to support growth at 18 degrees C. Consistent with its design, the R194L mutant hydrolyzes GTP at a reduced rate relative to wild-type Srp54 in enzymatic assays on immunoprecipitated proteins. In strains that also contain wild-type srp54, this mutant protein, as well as others designed to be locked in a GTP-bound conformation, exhibits temperature-dependent dominant inhibitory effects on growth, while a mutant predicted to be GDP locked does not interfere with the function of the wild-type protein. These results form the basis of a simple model for the role of GTP hydrolysis by Srp54 during the SRP cycle.     

An amphiphilic region in the cytoplasmic domain of KdpD is recognized by the signal recognition particle and targeted to the Escherichia coli membrane

The sensor protein KdpD of Escherichia coli is composed of a large N-terminal hydrophilic region (aa 1–400), four transmembrane regions (aa 401–498) and a large hydrophilic region (aa 499–894) at the C-terminus. KdpD requires the signal recognition particle (SRP) for its targeting to the membrane. Deletions within KdpD show that the first 50 residues are required for SRP-driven membrane insertion. A fusion protein of the green fluorescent protein (GFP) with KdpD is found localized at the membrane only when SRP is present. The membrane targeting of GFP was not observed when the first 50 KdpD residues were deleted. A truncated mutant of KdpD containing only the first 25 amino acids fused to GFP lost its ability to specifically interact with SRP, whereas a specific interaction between SRP and the first 48 amino acids of KdpD fused to GFP was confirmed by pull-down experiments. Conclusively, a small amphiphilic region of 27 residues within the amino-terminal domain of KdpD (aa 22–48) is recognized by SRP and targets the protein to the membrane. This shows that membrane proteins with a large N-terminal region in the cytoplasm can be membrane-targeted early on to allow co-translational membrane insertion of their distant transmembrane regions.     

Functional analysis of the signal recognition particle in Escherichia coli by characterization of a temperature-sensitive ffh mutant

The Ffh protein of Escherichia coli is a 48-kDa polypeptide that is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP). Efforts to understand the function of Ffh in bacteria have depended largely on the use of E. coli strains that allow depletion of the wild-type gene product. As an alternative approach to studying Ffh, a temperature-sensitive ffh mutant was isolated. The ffh-10(Ts) mutation results in two amino acid changes in conserved regions of the Ffh protein, and characterization of the mutant revealed that the cells rapidly lose viability at the nonpermissive temperature of 42 degrees C as well as show reduced growth at the permissive temperature of 30 degrees C. While the ffh mutant is defective in insertion of inner membrane proteins, the export of proteins with cleavable signal sequences is not impaired. The mutant also shows elevated expression of heat shock proteins and accumulates insoluble proteins, especially at 42 degrees C. It was further observed that the temperature sensitivity of the ffh mutant was suppressed by overproduction of 4.5S RNA, the RNA component of the bacterial SRP, by stabilizing the thermolabile protein. Collectively, these results are consistent with a model in which Ffh is required only for localization of proteins integral to the cytoplasmic membrane and suggest new genetic approaches to the study of how the structure of the SRP contributes to its function.     

Signals for the incorporation and orientation of cytochrome P450 in the endoplasmic reticulum membrane

Cytochrome P450b is an integral membrane protein of the rat hepatocyte endoplasmic reticulum (ER) which is cotranslationally inserted into the membrane but remains largely exposed on its cytoplasmic surface. The extreme hydrophobicity of the amino-terminal portion of P450b suggests that it not only serves to initiate the cotranslational insertion of the nascent polypeptide but that it also halts translocation of downstream portions into the lumen of the ER and anchors the mature protein in the membrane. In an in vitro system, we studied the cotranslational insertion into ER membranes of the normal P450b polypeptide and of various deletion variants and chimeric proteins that contain portion of P450b linked to segments of pregrowth hormone or bovine opsin. The results directly established that the amino-terminal 20 residues of P450b function as a combined insertion-halt-transfer signal. Evidence was also obtained that suggests that during the early stages of insertion, this signal enters the membrane in a loop configuration since, when the amino-terminal hydrophobic segment was placed immediately before a signal peptide cleavage site, cleavage by the luminally located signal peptidase took place. After entering the membrane, the P450b signal, however, appeared to be capable of reorienting within the membrane since a bovine opsin peptide segment linked to the amino terminus of the signal became translocated into the microsomal lumen. It was also found that, in addition to the amino-terminal combined insertion-halt-transfer signal, only one other segment within the P450b polypeptide, located between residues 167 and 185, could serve as a halt-transfer signal and membrane-anchoring domain. This segment was shown to prevent translocation of downstream sequences when the amino-terminal combined signal was replaced by the conventional cleavable insertion signal of a secretory protein.     

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.