The membrane-spanning segment of invariant chain (I gamma) contains a potentially cleavable signal sequence

The human invariant chain (I gamma) of class II histocompatibility antigens spans the membrane of the endoplasmic reticulum once. It exposes a small amino-terminal domain on the cytoplasmic side and a carboxy-terminal, glycosylated domain on the exoplasmic side of the membrane. When the exoplasmic domain of I gamma is replaced by the cytoplasmic protein chloramphenicol acetyltransferase (CAT), CAT becomes the exoplasmic, glycosylated domain of the resulting membrane protein I gamma CAT. Deletion of the hydrophilic cytoplasmic domain from I gamma CAT gives rise to a secreted protein from which an amino-terminal segment is cleaved, most likely by signal peptidase. We conclude that the membrane-spanning region of I gamma contains a signal sequence in its amino-terminal half and that hydrophilic residues at the amino-terminal end of a signal sequence can determine cleavage by signal peptidase.     

Signal peptidase can cleave inside a polytopic membrane protein

The signal peptides of most proteins targeted to the endoplasmic reticulum are specifically cleaved by signal peptidase. Although potential cleavage sites occur frequently in polytopic proteins after membrane-spanning segments, processing is restricted to the first hydrophobic domain, suggesting that signal peptidase might not have access to subsequently translocated, internal domains. To test this hypothesis, we replaced the third transmembrane segment of an artificial threefold membrane-spanning protein by a sequence which is normally an amino-terminal signal. Upon in vitro translation and insertion into microsomes, efficient cleavage at this sequence was observed, thus demonstrating the ability of signal peptidase to cleave within polytopic membrane proteins.     

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.     

SecY, a multispanning integral membrane protein, contains a potential leader peptidase cleavage site.

SecY is an Escherichia coli integral membrane protein required for efficient translocation of other proteins across the cytoplasmic membrane; it is embedded in this membrane by the 10 transmembrane segments. Among several SecY-alkaline phosphatase (PhoA) fusion proteins that we constructed previously, SecY-PhoA fusion 3-3, in which PhoA is fused to the third periplasmic region of SecY just after the fifth transmembrane segment, was found to be subject to rapid proteolytic processing in vivo. Both the SecY and PhoA products of this cleavage have been identified immunologically. In contrast, cleavage of SecY-PhoA 3-3 was barely observed in a lep mutant with a temperature-sensitive leader peptidase. The full-length fusion protein accumulated in this mutant was cleaved in vitro by the purified leader peptidase. A sequence Ala-202-Ile-Ala located near the proposed interface between transmembrane segment 5 and periplasmic domain 3 of SecY was found to be responsible for the recognition and cleavage by the leader peptidase, since a mutated fusion protein with Phe-Ile-Phe at this position was no longer cleaved even in the wild-type cells. These results indicate that SecY contains a potential leader peptidase cleavage site that undergoes cleavage if the PhoA sequence is attached carboxy terminally. Thus, transmembrane segment 5 of SecY can fulfill both of the two important functions of the signal peptide, translocation and cleavage, although the latter function is cryptic in the normal SecY protein.     

Phosphatidylinositol linkage of a truncated form of the platelet-derived growth factor receptor

The platelet-derived growth factor (PDGF) receptor is usually anchored to the plasma membrane through a membrane-spanning hydrophobic amino acid sequence that splits the molecule into two approximately equal pieces, an amino-terminal external domain that contains the binding site for PDGF and a carboxyl-terminal cytoplasmic domain that includes the tyrosine kinase coding sequences. Here we report the expression of a truncated PDGF receptor that consists of the extracellular domain without the transmembrane and cytoplasmic domains. Unexpectedly, this form of the receptor that lacks a hydrophobic membrane-anchoring sequence was bound to the membrane and was not secreted into the culture media. Conventional methods to dissociate noncovalent protein-protein interactions failed to release the protein from the membrane. When the transmembrane and cytoplasmic sequences were artificially deleted from the PDGF receptor, the truncated extracellular domain was anchored to the membrane through phospholipids and could be released by phospholipase C treatment. This truncated form of the receptor bound PDGF with an affinity 5-20-fold lower than the full-length receptor.     

The role of the polar, carboxyl-terminal domain of Escherichia coli leader peptidase in its translocation across the plasma membrane

Leader peptidase, an integral membrane protein of Escherichia coli, is made without a cleavable leader sequence. It has 323 amino acid residues and spans the plasma membrane with a small amino-terminal domain exposed to the cytoplasm and a large, carboxyl-terminal domain exposed to the periplasm. We have investigated which regions of leader peptidase are necessary for its assembly across the membrane. Deletions were made in the carboxyl-terminal domain of leader peptidase, removing residues 141-222, 142-323, or 222-323. Protease accessibility was used to determine whether the polar, carboxyl-terminal domains of these truncated leader peptidases were translocated across the membrane. The removal of either residues 222-323 (the extreme carboxyl terminus) or residues 141-222 does not prevent leader peptidase membrane assembly. However, leader peptidase lacking both regions, i.e. amino acid residues 142-323, cannot translocate the remaining portion of its carboxyl terminus across the membrane. Our data suggest that the polar, periplasmic domain of leader peptidase contains information which is needed for membrane assembly.     

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.     

Modified enterotoxin signal sequences increase secretion level of the recombinant human epidermal growth factor in Escherichia coli

Amino acid substitutions were made in the heat-labile enterotoxin signal sequence of Escherichia coli by recombinant DNA techniques, and their influence on the secretion of recombinant human epidermal growth factor by E. coli was examined. The heat-labile enterotoxin signal sequence is an amino-terminal extension of the octadecapeptide chain and is comprised of three distinct regions: a positively charged amino-terminal region, a central hydrophobic region, and a carboxyl-terminal region with the cleavage site recognized by the signal peptidase. Some alterations in the signal sequence caused a 1.5-3.5-fold increase in the secretion of recombinant human epidermal growth factor. These were the introduction of: (i) polar and small residues into the carboxyl-terminal region (replacement of Pro-1 Leu-3 with Asn-Ala or Ser-Ala), which may give a favorable structure for the recognition and cleavage by the signal peptidase; and (ii) a polar residue into the central hydrophobic region (replacement of Ile-9 with Ser), which may cause an increase of the affinity to the cytoplasmic membrane. In the latter case, a large amount of the unprocessed "precursor" was accumulated. The combination of these modifications, however, did not work additively. An increase in the amino-terminal positive charge (insertion of Lys) had no effect on secretion. These results prove that the level of protein secretion is greatly dependent on the polarity of the carboxyl-terminal region and the hydrophobicity and/or the amphiphilicity of the central region. Moreover, the overall balance of the physicochemical properties of respective regions is important.     

Distinct domains of an oligotopic membrane protein are Sec-dependent and Sec-independent for membrane insertion.

Leader peptidase of Escherichia coli spans the plasma membrane twice with its amino terminus on the periplasmic surface of the membrane and its large carboxyl-terminal domain protruding into the periplasm. To monitor the transfer of the amino terminus of leader peptidase to the periplasm, we have constructed a fusion protein between the 18-residue amino-terminal periplasmic domain of Pf3 bacteriophage coat protein and the beginning of leader peptidase. We find that neither the SecA or SecY proteins nor a transmembrane electrochemical potential is required for insertion of the amino terminus, while the transfer of the carboxyl-terminal domain of leader peptidase has these requirements. The first 35 residues of leader peptidase, which include the first hydrophobic domain and the carboxyl-terminal positively charged cluster, are sufficient to insert the amino terminus. When positively charged residues are introduced before the first transmembrane segment, translocation of the amino terminus is abolished. These studies in protein membrane topogenesis, showing that there are different requirements for amino and carboxyl termini insertion, indicate that multiple mechanisms exist even within the same protein.     

Use of phoA fusions to study the topology of the Escherichia coli inner membrane protein leader peptidase.

A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of proteolytic studies. Here, a collection of alkaline phosphatase fusions to leader peptidase is described. Fusions to the periplasmic domain of this protein exhibit high alkaline phosphatase activity, while fusions to the cytoplasmic domain exhibit low activity. Elements within the cytoplasmic domain are necessary to stably anchor alkaline phosphatase in the cytoplasm. The amino-terminal hydrophobic segment of leader peptidase acts as a weak export signal for alkaline phosphatase. However, when this segment is preceded by four lysines, it acts as a highly efficient export signal. The coherence of in vitro studies with alkaline phosphatase fusion analysis of the topology of leader peptidase further indicates the utility of this genetic approach to membrane protein structure and insertion.     

The amino-terminal domain of the lamin B receptor is a nuclear envelope targeting signal

The lamin B receptor (LBR) is a polytopic protein of the inner nuclear membrane. It is synthesized without a cleavable amino-terminal signal sequence and composed of a nucleoplasmic amino-terminal domain of 204 amino acids followed by a hydrophobic domain with eight putative transmembrane segments. To identify a nuclear envelope targeting signal, we have examined the cellular localization by immunofluorescence microscopy of chicken LBR, its amino-terminal domain and chimeric proteins transiently expressed in transfected COS-7. Full- length LBR was targeted to the nuclear envelope. The amino-terminal domain, without any transmembrane segments, was transported to the nucleus but excluded from the nucleolus. When the amino-terminal domain of LBR was fused to the amino-terminal side of a transmembrane segment of a type II integral membrane protein of the ER/plasma membrane, the chimeric protein was targeted to the nuclear envelope, likely the inner nuclear membrane. When the amino-terminal domain was deleted from LBR and replaced by alpha-globin, the chimeric protein was retained in the ER. These findings demonstrate that the amino-terminal domain of LBR is targeted to the nucleus after synthesis in the cytoplasm and that this polypeptide can function as a nuclear envelope targeting signal when located at the amino terminus of a type II integral membrane protein synthesized on the ER.     

Epstein-Barr virus recombinant molecular genetic analysis of the LMP1 amino-terminal cytoplasmic domain reveals a probable structural role, with no component essential for primary B-lymphocyte growth transformation.

Previous recombinant Epstein-Barr virus molecular genetic experiments with specifically mutated LMP1 genes indicate that LMP1 is essential for primary B-lymphocyte growth transformation and that the amino-terminal cytoplasmic and first transmembrane domains are together an important mediator of transformation. EBV recombinants with specific deletions in the amino-terminal cytoplasmic domain have now been constructed and tested for the ability to growth transform primary B lymphocytes into lymphoblastoid cell lines. Surprisingly, deletion of DNA encoding EHDLER or GPPLSSS from the full LMP1 amino-terminal cytoplasmic domain (MEHDLERGPPGPRRPPRGPPLSSS) had no discernible effect on primary B-lymphocyte transformation. These two motifs distinguish the LMP1 amino-terminal cytoplasmic domain from other arginine-rich membrane proximal sequences that anchor hydrophobic transmembrane domains. Two deletions which included the ERGPPGPRRPPR motif adversely affected but did not prevent transformation. This arginine- and proline-rich sequence is probably important in anchoring the first transmembrane domain in the plasma membrane, since these mutated LMP1s had altered stability and cell membrane localization. The finding that overlapping deletions of the entire amino-terminal cytoplasmic domain do not ablate transformation is most consistent with a model postulating that the transmembrane and carboxyl-terminal cytoplasmic domains are the likely biochemical effectors of transformation.     

Leader peptidase

The Escherichia coli leader peptidase has been vital for unravelling problems in membrane assembly and protein export. The role of this essential peptidase is to remove amino-terminal leader peptides from exported proteins after they have crossed the plasma membrane. Strikingly, almost all periplasmic proteins, many outer membrane proteins, and a few inner membrane proteins are made with cleavable leader peptides that are removed by this peptidase. This enzyme of 323 amino acid residues spans the membrane twice, with its large carboxyl-terminal domain protruding into the periplasm. Recent discoveries show that its membrane orientation is controlled by positively charged residues that border (on the cytosolic side) the transmembrane segments. Cleavable pre-proteins must have small residues at -1 and a small or aliphatic residue at -3 (with respect to the cleavage site). Leader peptidase does not require a histidine or cysteine amino acid for catalysis. Interestingly, serine 90 and aspartic acid 153 are essential for catalysis and are also conserved in a mitochondrial leader peptidase, which is 30.7% homologous with the bacterial enzyme over a 101-residue stretch.     

Signals and structural features involved in integral membrane protein targeting to the inner nuclear membrane

We have examined transfected cells by immunofluorescence microscopy to determine the signals and structural features required for the targeting of integral membrane proteins to the inner nuclear membrane. Lamin B receptor (LBR) is a resident protein of the nuclear envelope inner membrane that has a nucleoplasmic, amino-terminal domain and a carboxyl-terminal domain with eight putative transmembrane segments. The amino-terminal domain of LBR can target both a cytosolic protein to the nucleus and a type II integral protein to the inner nuclear membrane. Neither a nuclear localization signal (NLS) of a soluble protein, nor full-length histone H1, can target an integral protein to the inner nuclear membrane although they can target cytosolic proteins to the nucleus. The addition of an NLS to a protein normally located in the inner nuclear membrane, however, does not inhibit its targeting. When the amino-terminal domain of LBR is increased in size from approximately 22.5 to approximately 70 kD, the chimeric protein cannot reach the inner nuclear membrane. The carboxyl-terminal domain of LBR, separated from the amino-terminal domain, also concentrates in the inner nuclear membrane, demonstrating two nonoverlapping targeting signals in this protein. Signals and structural features required for the inner nuclear membrane targeting of proteins are distinct from those involved in targeting soluble polypeptides to the nucleoplasm. The structure of the nucleocytoplasmic domain of an inner nuclear membrane protein also influences targeting, possibly because of size constraints dictated by the lateral channels of the nuclear pore complexes.     

Insertion of a signal peptide-derived hydrophobic segment into the mature domain of OmpC, an outer membrane protein, does not interfere with the export of the following polypeptide chain across the cytoplasmic membrane of E. coli

The effects of a hydrophobic peptide segment inserted into the amino-terminal region of the mature domain of OmpC, an outer membrane protein, on its translocation across the cytoplasmic membrane was studied. Both the intact OmpC and central domain-deleted OmpC were examined. The hydrophobic segment was derived from the signal peptide of OmpF. Secretory translocation across the cytoplasmic membrane was examined by means of proteinase K treatment. Four monoclonal antibodies that recognize different regions of OmpC were used to characterize proteinase K-resistant fragments. Insertion of the hydrophobic segment did not appreciably prevent the translocation of these proteins across the cytoplasmic membrane, larger parts of them being found as mature forms, which were mostly localized outside the cytoplasmic membrane. Circumstantial evidence supports the view, on the other hand, that the inserted hydrophobic domain was retained in the cytoplasmic membrane. It is concluded, therefore, that the hydrophobic segment, although it is not exported across the cytoplasmic membrane, does not prevent the secretion of the following polypeptide chain. The secretion was dependent on the amino-terminal signal peptide. Insertion of positive charges immediately after the hydrophobic segment resulted in suppression of the translocation. Based on these results possible mechanisms by which the secretion of the polypeptide chain after the hydrophobic segment are discussed.     

The role of the membrane-spanning domain of type I signal peptidases in substrate cleavage site selection

Type I signal peptidase (SPase I) catalyzes the cleavage of the amino-terminal signal sequences from preproteins destined for cell export. Preproteins contain a signal sequence with a positively charged n-region, a hydrophobic h-region, and a neutral but polar c-region. Despite having no distinct consensus sequence other than a commonly found c-region "Ala-X-Ala" motif preceding the cleavage site, signal sequences are recognized by SPase I with high fidelity. Remarkably, other potential Ala-X-Ala sites are not cleaved within the preprotein. One hypothesis is that the source of this fidelity is due to the anchoring of both the SPase I enzyme (by way of its transmembrane segment) and the preprotein substrate (by the h-region in the signal sequence) in the membrane. This limits the enzyme-substrate interactions such that cleavage occurs at only one site. In this work we have, for the first time, successfully isolated Bacillus subtilis type I signal peptidase (SipS) and a truncated version lacking the transmembrane domain (SipS-P2). With purified full-length as well as truncated constructs of both B. subtilis and Escherichia coli (Lep) SPase I, in vitro specificity studies indicate that the transmembrane domains of either enzyme are not important determinants of in vitro cleavage fidelity, since enzyme constructs lacking them reveal no alternate site processing of pro-OmpA nuclease A substrate. In addition, experiments with mutant pro-OmpA nuclease A substrate constructs indicate that the h-region of the signal peptide is also not critical for substrate specificity. In contrast, certain mutants in the c-region of the signal peptide result in alternate site cleavage by both Lep and SipS enzymes.     

The Disabled 1 Phosphotyrosine-Binding Domain Binds to the Internalization Signals of Transmembrane Glycoproteins and to Phospholipids

Disabled gene products are important for nervous system development in drosophila and mammals. In mice, the Dab1 protein is thought to function downstream of the extracellular protein Reln during neuronal positioning. The structures of Dab proteins suggest that they mediate protein-protein or protein-membrane docking functions. Here we show that the amino-terminal phosphotyrosine-binding (PTB) domain of Dab1 binds to the transmembrane glycoproteins of the amyloid precursor protein (APP) and low-density lipoprotein receptor families and the cytoplasmic signaling protein Ship. Dab1 associates with the APP cytoplasmic domain in transfected cells and is coexpressed with APP in hippocampal neurons. Screening of a set of altered peptide sequences showed that the sequence GYXNPXY present in APP family members is an optimal binding sequence, with approximately 0.5 microM affinity. Unlike other PTB domains, the Dab1 PTB does not bind to tyrosine-phosphorylated peptide ligands. The PTB domain also binds specifically to phospholipid bilayers containing phosphatidylinositol 4P (PtdIns4P) or PtdIns4,5P2 in a manner that does not interfere with protein binding. We propose that the PTB domain permits Dab1 to bind specifically to transmembrane proteins containing an NPXY internalization signal.     

The internal signal sequence of Escherichia coli leader peptidase is necessary, but not sufficient, for its rapid membrane assembly

Leader peptidase of Escherichia coli, a protein of 323 residues, has three hydrophobic domains. The first, residues 1-22, is the most apolar and is followed by a polar region (23-61) which faces the cytoplasm. The second hydrophobic domain (residues 62-76) spans the membrane. The third hydrophobic domain, which has a minimal apolar character, and the polar, carboxyl-terminal two-thirds of the protein are exposed to the periplasm. Deletion of either the amino terminus (residues 4-50) or the third hydrophobic region (residues 83-98) has almost no effect on the rate of leader peptidase membrane assembly, while the second hydrophobic domain is essential for insertion (Dalbey, R., and Wickner, W. (1987) Science 235, 783-787). To further define the roles of these domains, we have replaced the normal, cleaved leader sequence of pro-OmpA and M13 procoat with regions containing either the first or second apolar domain of leader peptidase. The second apolar domain supports the translocation of OmpA or coat protein across the plasma membrane, establishing its identity as an internal, uncleaved signal sequence. In addition to this sequence, we now find that leader peptidase needs either the amino-terminal domain or the third hydrophobic domain to permit its rapid membrane assembly. These results show that, although a signal sequence is necessary for rapid membrane assembly of leader peptidase, it is not sufficient.     

Role of the TonB amino terminus in energy transduction between membranes.

Escherichia coli TonB protein is an energy transducer, coupling cytoplasmic membrane energy to active transport of vitamin B12 and iron-siderophores across the outer membrane. TonB is anchored in the cytoplasmic membrane by its hydrophobic amino terminus, with the remainder occupying the periplasmic space. In this report we establish several functions for the hydrophobic amino terminus of TonB. A G-26-->D substitution in the amino terminus prevents export of TonB, suggesting that the amino terminus contains an export signal for proper localization of TonB within the cell envelope. Substitution of the first membrane-spanning domain of the cytoplasmic membrane protein TetA for the TonB amino terminus eliminates TonB activity without altering TonB export, suggesting that the amino terminus contains sequence-specific information. Detectable TonB cross-linking to ExbB is also prevented, suggesting that the two proteins interact primarily through their transmembrane domains. In vivo cleavage of the amino terminus of TonB carrying an engineered leader peptidase cleavage site eliminates (i) TonB activity, (ii) detectable interaction with a membrane fraction having a density intermediate to those of the cytoplasmic and outer membranes, and (iii) cross-linking to ExbB. In contrast, the amino terminus is not required for cross-linking to other proteins with which TonB can form complexes, including FepA. Additionally, although the amino terminus clearly is a membrane anchor, it is not the only means by which TonB associates with the cytoplasmic membrane. TonB lacking its amino-terminal membrane anchor still remains largely associated with the cytoplasmic membrane.