Signals and mechanisms for protein retention in the endoplasmic reticulum

After their co-translational insertion into the ER lumen or the ER membrane, most proteins are transported via the Golgi apparatus downstream on the secretory pathway while a few protein species are retained in the ER. Polypeptide retention in the ER is either signal-independent or depends on specific retention signals encoded by the primary sequence of the polypeptide. A first category, i.e. the newly synthesized polypeptides that are unable to reach their final conformation, are retained in the ER where this quality control generally results in their degradation. A second category, namely the ER-resident proteins escape the bulk flow of secretion due to the presence of a specific N- or C-terminal signal that interacts with integral membrane or soluble receptors. ER retention of soluble proteins mediated by either KDEL, HDEL or related sequences and membrane receptors has been relatively well characterized in plants. Recent efforts has been relatively well characterized in plants. Recent efforts have aimed at a characterization of the retention signal(s) of type I membrane proteins in the plant ER.     

Nucleotide sequence of the gene for the ferrienterochelin receptor FepA in Escherichia coli. Homology among outer membrane receptors that interact with TonB

We have determined the nucleotide sequence of the Escherichia coli fepA gene, which codes for the outer membrane receptor for ferrienterochelin and colicins B and D. The predicted FepA polypeptide has a molecular weight of 79,908 and consists of 723 amino acids. A 22-amino acid leader or signal peptide preceded the mature protein. With respect to overall composition, hydropathy, net charge and distribution of nonpolar segments, the FepA polypeptide was typical of other E. coli outer membrane proteins, except that FepA contained 2 cysteine residues. Comparison of the deduced amino acid sequence of FepA with that of three other TonB-dependent receptors (BtuB, FhuA, and IutA) revealed only a few regions of sequence homology; one of these included the amino-termini. An amino acid substitution within the conserved amino-terminal region of BtuB resulted in production of a receptor that had normal binding functions but was incapable of energy-dependent transport of vitamin B12. This result suggests that the amino-terminal end of these four polypeptides is involved in interaction with the TonB protein or another step of energy transduction. Three other regions of homology were shared among the four proteins: one near residues 50 to 70, another at about residue 100 to 140, and the last between 20 and 40 amino acid residues from the carboxyl terminus. The function of these three regions remains speculative.     

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.     

Charged residues are major determinants of the transmembrane orientation of a signal-anchor sequence

Uncleaved signal-anchor sequences of membrane proteins inserted into the endoplasmic reticulum initiate the translocation of either the amino-terminal or the carboxyl-terminal polypeptide segment across the bilayer. Which topology is acquired is not determined by the apolar segment of the signal but rather by the hydrophilic sequences flanking it. To study the role of charged residues in determining the membrane topology, the insertion of mutants of the asialoglycoprotein receptor H1, a single-spanning protein with a cytoplasmic amino terminus, was analyzed in transfected COS-7 cells. When the charged amino acids flanking the hydrophobic signal were mutated to residues of opposite charge, half the polypeptides inserted with the inverted orientation. When, in addition, the amino-terminal domain of the mutant protein was truncated, approximately 90% of the polypeptides acquired the inverted topology. The transmembrane orientation appears to be primarily determined by the charges flanking the signal sequence but is modulated by the domains to be translocated.     

Molecular dissection of the NH2-terminal signal/anchor sequence of rat dipeptidyl peptidase IV

Dipeptidyl peptidase IV (DPPIV) is a membrane glycoprotein with a type II orientation in the plasma membrane. As shown in a cell-free translation system, the amino-terminal 34 amino acids of rat DPPIV are involved in translocating nascent polypeptide across the membrane of microsomes and in anchoring the translocated polypeptide in the microsomal membrane. The amino-terminal sequence performing this dual function is composed of: a central hydrophobic core of 22 amino acid residues; 6 amino-terminal residues preceding the hydrophobic core (MKTPWK); and 6 residues following the hydrophobic core. The six residues preceding the hydrophobic core are exposed on the outside (cytoplasmic side) of the microsomal membrane. Site-directed mutagenesis studies show that deletion of this cytoplasmic domain, excluding the amino-terminal initiating methionine, does not affect translocation of nascent DPPIV polypeptide, but does affect significantly anchoring of the translocated polypeptide in the microsomal membrane. In contrast, changing the two cytoplasmic Lys to Glu residues or shortening of the hydrophobic core from 22 to 15 residues or converting the last 11e of the shortened hydrophobic core into Ala affects neither translocation across nor anchoring of the DPPIV polypeptide in the microsomal membrane. These and other structural features of the DPPIV amino-terminal signal-anchor sequences are discussed along with other types of sequences for their role in targeting nascent polypeptides to the RER.     

The signal recognition particle receptor alpha subunit assembles co-translationally on the endoplasmic reticulum membrane during an mRNA-encoded translation pause in vitro.

Many proteins, including the alpha subunit of the signal recognition particle receptor (SR alpha), are targeted within the cell by poorly defined mechanisms. A 140 residue N-terminal domain of SR alpha targets and anchors the polypeptide to the endoplasmic reticulum membrane by a mechanism independent of the pathway involving the signal recognition particle. To investigate the mechanism of membrane anchoring, translation pause sites on the SR alpha mRNA were used to examine the targeting of translation intermediates. A strong pause site at nucleotide 507 of the mRNA open reading frame corresponded with the shortest nascent SR alpha polypeptide able to assemble on membranes. An mRNA sequence at this pause site that resembles a class of viral -1 frameshift sequences caused translation pausing when transferred into another mRNA context. Site-directed mutagenesis of the mRNA greatly reduced translation pausing without altering the polypeptide sequence, demonstrating unambiguously a role for this mRNA sequence in translation pausing. SR alpha polypeptides synthesized from the non-pausing mRNA were impaired in co-translational membrane anchoring. Furthermore, co-translational membrane assembly of SR alpha appears to anchor polysomes translating SR alpha to membranes.     

An internalized amino-terminal signal sequence retains full activity in vivo but not in vitro

Internalization of the signal sequence of the vesicular stomatitis virus glycoprotein was accomplished by extending the amino-terminal coding sequence with sequences derived from pBR322. Such constructs were then expressed in eukaryotic cells. It was found that amino-terminal extensions consisting of 20, 61, or 102 amino acids totally unrelated to any signal peptide affected neither the function nor cleavage of the signal sequence in vivo. Subsequent transport of the glycoprotein was also not affected. Although the internalized signals functioned with wild-type efficiency in vivo, membrane insertion in vitro (as determined by proteolysis protection assays), signal cleavage, and glycosylation were only achieved when the amino-terminal presequences were short.     

Glycosylation can influence topogenesis of membrane proteins and reveals dynamic reorientation of nascent polypeptides within the translocon.

The topology of multispanning membrane proteins in the mammalian endoplasmic reticulum is thought to be dictated primarily by the first hydrophobic sequence. We analyzed the in vivo insertion of a series of chimeric model proteins containing two conflicting signal sequences, i.e., an NH(2)-terminal and an internal signal, each of which normally directs translocation of its COOH-terminal end. When the signals were separated by more than 60 residues, linear insertion with the second signal acting as a stop-transfer sequence was observed. With shorter spacers, an increasing fraction of proteins inserted with a translocated COOH terminus as dictated by the second signal. Whether this resulted from membrane targeting via the second signal was tested by measuring the targeting efficiency of NH(2)-terminal signals followed by polypeptides of different lengths. The results show that targeting is mediated predominantly by the first signal in a protein. Most importantly, we discovered that glycosylation within the spacer sequence affects protein orientation. This indicates that the nascent polypeptide can reorient within the translocation machinery, a process that is blocked by glycosylation. Thus, topogenesis of membrane proteins is a dynamic process in which topogenic information of closely spaced signal and transmembrane sequences is integrated.     

Deletion analysis of the internal signal-anchor domain of the human asialoglycoprotein receptor H1.

The human asialoglycoprotein receptor H1 is a single-spanning membrane protein with the amino terminus facing the cytoplasm and the carboxy terminus exposed on the exoplasmic side of the plasma membrane. It has been shown earlier that the transmembrane segment, residues 38-65, functions as an internal signal directing protein synthesis to the endoplasmic reticulum and initiating membrane insertion. This process is co-translational and mediated by signal recognition particle (SRP). To identify subsegments within this region containing the signal information, we prepared deletion mutants at the level of the cDNA and analysed them in a wheat germ in vitro translation system with microsomes as the target membrane. Insertion and membrane anchoring were judged by the glycosylation of the protein, its resistance to exogenous protease and the extent to which it can be extracted from the microsomes by alkaline treatment. It was found that very small deletions already reduce the stability of membrane anchoring. However, nearly half of the transmembrane domain can be deleted, both from the amino-terminal and from the carboxy-terminal side, without completely abolishing membrane insertion. Several mutants, although not inserted, still interact with SRP. The results support the notion that the main feature of a signal sequence is a hydrophobic stretch of sufficient length (10-12 residues in our sequence), and indicate that recognition by SRP is not sufficient for membrane insertion.     

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.     

In vivo mitochondrial import. A comparison of leader sequence charge and structural relationships with the in vitro model resulting in evidence for co-translational import

The positive charges and structural properties of the mitochondrial leader sequence of aldehyde dehydrogenase have been extensively studied in vitro. The results of these studies showed that increasing the helicity of this leader would compensate for reduced import from positive charge substitutions of arginine with glutamine or the insertion of negative charged residues made in the native leader. In this in vivo study, utilizing the green fluorescent protein (GFP) as a passenger protein, import results showed the opposite effect with respect to helicity, but the results from mutations made within the native leader sequence were consistent between the in vitro and in vivo experiments. Leader mutations that reduced the efficiency of import resulted in a cytosolic accumulation of a truncated GFP chimera that was fluorescent but devoid of a mitochondrial leader. The native leader efficiently imported before GFP could achieve a stable, import-incompetent structure, suggesting that import was coupled with translation. As a test for a co-translational mechanism, a chimera of GFP that contained the native leader of aldehyde dehydrogenase attached at the N terminus and a C-terminal endoplasmic reticulum targeting signal attached to the C terminus of GFP was constructed. This chimera was localized exclusively to mitochondria. The import result with the dual signal chimera provides support for a co-translational mitochondrial import pathway.     

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.     

N-terminal amino acid sequences and amino acid compositions of the Spirochaeta aurantia flagellar filament polypeptides.

The amino-terminal sequences and amino acid compositions of the three major and two minor polypeptides constituting the filaments of Spirochaeta aurantia periplasmic flagella were determined. The amino-terminal sequence of the major 37.5-kDa outer layer polypeptide is identical to the sequence downstream of the proposed signal peptide of the protein encoded by the S. aurantia flaA gene. However, the amino acid composition of the 37.5-kDa polypeptide is not in agreement with that inferred from the sequence of flaA. The 34- and 31.5-kDa major filament core polypeptides and the 33- and 32-kDa minor core polypeptides show a striking similarity to each other, and the amino-terminal sequences of these core polypeptides show extensive identity with homologous proteins from members of other genera of spirochetes. An additional 36-kDa minor polypeptide that occurs occasionally in preparations of S. aurantia periplasmic flagella appears to be mixed with the 37.5-kDa outer layer polypeptide or a degradation product of this polypeptide.     

The amino terminus of GLUT4 functions as an internalization motif but not an intracellular retention signal when substituted for the transferrin receptor cytoplasmic domain

Previous studies have demonstrated that the amino-terminal cytoplasmic domain of GLUT4 contains a phenylalanine-based targeting motif that determines its steady state distribution between the surface and the interior of cells (Piper, R. C., C. Tai, P. Kuleza, S. Pang, D. Warnock, J. Baenziger, J. W. Slot, H. J. Geuze, C. Puri, and D. E. James. 1993. J. Cell Biol. 121:1221). To directly measure the effect that the GLUT4 amino terminus has on internalization and subsequent recycling back to the cell surface, we constructed chimeras in which this sequence was substituted for the amino-terminal cytoplasmic domain of the human transferrin receptor. The chimeras were stably transfected into Chinese hamster ovary cells and their endocytic behavior characterized. The GLUT4-transferrin receptor chimera was recycled back to the cell surface with a rate similar to the transferrin receptor, indicating that the GLUT4 sequence was not promoting intracellular retention of the chimera. The GLUT4-transferrin receptor chimera was internalized at half the rate of the transferrin receptor. Substitution of an alanine for phenylalanine at position 5 slowed internalization of the chimera by twofold, to a level characteristic of bulk membrane internalization. However, substitution of a tyrosine increased the rate of internalization to the level of the transferrin receptor. Neither of these substitutions significantly altered the rate at which the chimeras were recycled back to the cell surface. These results demonstrate that the major function of the GLUT4 amino-terminal domain is to promote the effective internalization of the protein from the cell surface, via a functional phenylalanine-based internalization motif, rather than retention of the transporter within intracellular structures.     

Transport of proteins in eukaryotic cells: more questions ahead

Some newly synthesized proteins contain signals that direct their transport to their final location within or outside of the cell. Targeting signals are recognized by specific protein receptors located either in the cytoplasm or in the membrane of the target organelle. Specific membrane protein complexes are involved in insertion and translocation of polypeptides across the membranes. Often, additional targeting signals are required for a polypeptide to be further transported to its site of function. In this review, we will describe the trafficking of proteins to various cellular organelles (nucleus, chloroplasts, mitochondria, peroxisomes) with emphasis on transport to and through the secretory pathway.     

A hydrophobic region locating at the center of fibroblast growth factor-9 is crucial for its secretion.

Fibroblast growth factor (FGF)-9 is a glycosylated neurotrophic polypeptide highly expressed in brain. The mechanism for its secretion from expressing cells is unclear, because its primary structure lacks a cleavable signal sequence. We, therefore, investigated the mechanism and structural requirements for secretion of FGF-9. As with other secreted proteins, in vitro translation of FGF-9 was inhibited by signal recognition particle, which binds to the signal sequence. When translated in vitro, full-length FGF-9 was translocated into microsomes, glycosylated, and protected from trypsin digestion. By using various FGF-9 deletion mutants, we found that two hydrophobic domains, located at the N terminus and at the center of the FGF-9 primary structure, were crucial for translocation. Examination of various point mutants revealed that local hydrophobicity of the central hydrophobic domain, but not the N terminus, was crucial for translocation. Analogous results were obtained with respect to FGF-9 secretion from transfectant cells. Upon deletion of the complete sequence preceding it, the previously uncleavable hydrophobic domain appeared to serve as a cleavable signal sequence. Our results suggest that nascent FGF-9 polypeptides translocate into endoplasmic reticulum without peptide cleavage via a co-translational pathway in which both the N terminus and the central hydrophobic domain are important; thereafter, FGF-9 is glycosylated and secreted.     

Topogenesis of mitochondrial inner membrane uncoupling protein. Rerouting transmembrane segments to the soluble matrix compartment

Brown adipose tissue uncoupling protein (UCP), an integral polytopic protein of the mitochondrial inner membrane, is composed of at least six transmembrane segments whose net hydrophobic character derives from paired amphiphilic helices. The protein is synthesized in the cytoplasm as a polypeptide (307 amino acids) lacking a cleavable targeting (signal) peptide. Deletion mutagenesis and fusion protein constructions revealed the existence of at least two import signals: one lying between UCP precursor amino acids 13-105 and the other downstream of position 101. The former resulted in both targeting and membrane insertion of a fusion protein, whereas the latter targeted UCP 102-307 into the organelle but failed to result in membrane insertion. When a strong matrix-targeting signal derived from precarbamoyl phosphate synthetase was fused to UCP amino acids 169-307 or 52-307 (containing three and five transmembrane domains, respectively), the fusion proteins were efficiently imported to the soluble matrix compartment where correct signal cleavage took place. We suggest that assembly of UCP into the inner membrane follows a coordinate insertion pathway for integration and may use more than one signal sequence to achieve this. In this respect, it might share certain mechanistic features with the insertion of polytopic proteins into the endoplasmic reticulum. The data also suggest, however, that integration of the amino-terminal third of UCP into the inner membrane may be required to help or enhance insertion of the remaining UCP transmembrane domains.     

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.