Expression of the presenilin-like signal peptide peptidase (SPP) in mouse adult brain and during development

Recently, a new member of the presenilin family was identified as an aspartyl protease that cleaves signal peptides within hydrophobic domains, and was, therefore, named signal peptide peptidase (SPP). We isolated cDNAs coding for mouse and human orthologues of SPP. The human gene spans 55 kilobases on chromosome 20q11.21. The SPP-protein is encoded in mouse and man by 12 exons. The highly conserved intron/exon-structure in the SPP/presenilin family hints at a common precursor. Northern blot and in situ hybridization analysis revealed a widespread expression of SPP in many tissues. A distinct pattern of expression in the mature murine brain and during development indicates that SPP plays an important role in the establishment and maintenance of the nervous system. We prepared an antiserum against the carboxy-terminal domain of SPP, which is highly conserved between species. It reacted specifically, both in western blots and in immunocytochemical preparations, with SPP from various mammalian origins. The antiserum was used to demonstrate that SPP is oriented in the membrane of the endoplasmic reticulum with its carboxy-terminal tail extending into the cytosol.     

Intramembrane proteolysis and endoplasmic reticulum retention of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein is suggested to localize to the endoplasmic reticulum (ER) through a C-terminal hydrophobic region that acts as a membrane anchor for core protein and as a signal sequence for E1 protein. The signal sequence of core protein is further processed by signal peptide peptidase (SPP). We examined the regions of core protein responsible for ER retention and processing by SPP. Analysis of the intracellular localization of deletion mutants of HCV core protein revealed that not only the C-terminal signal-anchor sequence but also an upstream hydrophobic region from amino acid 128 to 151 is required for ER retention of core protein. Precise mutation analyses indicated that replacement of Leu(139), Val(140), and Leu(144) of core protein by Ala inhibited processing by SPP, but cleavage at the core-E1 junction by signal peptidase was maintained. Additionally, the processed E1 protein was translocated into the ER and glycosylated with high-mannose oligosaccharides. Core protein derived from the mutants was translocated into the nucleus in spite of the presence of the unprocessed C-terminal signal-anchor sequence. Although the direct association of core protein with a wild-type SPP was not observed, expression of a loss-of-function SPP mutant inhibited cleavage of the signal sequence by SPP and coimmunoprecipitation with unprocessed core protein. These results indicate that Leu(139), Val(140), and Leu(144) in core protein play crucial roles in the ER retention and SPP cleavage of HCV core protein.     

Retention of a type II surface membrane protein in the endoplasmic reticulum by the Lys-Asp-Glu-Leu sequence.

Soluble luminal proteins of the endoplasmic reticulum (ER) are known to be retained by a tetrapeptide retention signal, KDEL. We report in this communication that the KDEL sequence when appended to the carboxy terminus of a cell surface membrane protein, dipeptidyl peptidase IV (DPPIV), resulted in its retention in the endoplasmic reticulum of transfected Madin-Darby canine kidney cells as assessed by indirect immunofluorescence. Selective surface biotinylation revealed that about 90-95% of the expressed DPPIV was retained in the ER. Appendance of the sequence KDEV did not, however, result in ER retention, illustrating the functional specificity of the retention signal. The ER retention was not due to misfolding of the mutant protein, as the mutant proteins remained enzymatically active. Our data suggest that the KDEL receptor is able to recognize and recycle type II membrane proteins containing a carboxyl-terminal KDEL sequence and postulates the existence of such yet to be identified endogenous proteins.     

The eta isoform of protein kinase C is localized on rough endoplasmic reticulum.

The eta isoform of protein kinase C, isolated from a cDNA library of mouse skin, has unique tissue and cellular distributions. It is predominantly expressed in epithelia of the skin, digestive tract, and respiratory tract in close association with epithelial differentiation. We report here that this isoform is localized on the rough endoplasmic reticulum in transiently expressing COS1 cells and constitutively expressing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluorescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron microscopic examination, electron-dense signals were located on the rough endoplasmic reticulum and on the outer nuclear membrane which is continuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treatment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunoblotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions, all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localization signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chaim, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12:1304-1311, 1992).     

Peripheral membrane proteins of sarcoplasmic and endoplasmic reticulum. Comparison of carboxyl-terminal amino acid sequences

Peripheral endoplasmic reticulum membrane proteins residing in the lumen of the endoplasmic reticulum occupy the same space as other secreted proteins. The presence of a four amino acid salvage or retention signal (KDEL-COOH = Lys-Asp-Glu-Leu-COOH) at the carboxyl-terminal end of peripheral membrane proteins has been shown to represent a signal or an essential part of a signal for their retention within the endoplasmic reticulum membrane. In heart and skeletal muscle, a number of sarcoplasmic reticulum proteins have recently been identified which are peripheral membrane proteins. The high-affinity calcium-binding protein (55 kilodaltons (kDa] appears to conform to the above described mechanisms and contains the KDEL carboxyl-terminal tetrapeptide. Thyroid hormone binding protein is present in the sarcoplasmic reticulum, in addition to its endoplasmic reticulum location, and has a modified but related tetrapeptide sequence (RDEL = Arg-Asp-Glu-Leu), which also probably functions as the retention signal. Calsequestrin and a 53-kDa glycoprotein, two other peripheral membrane proteins residing in the lumen of the sarcoplasmic reticulum, do not contain the KDEL retention signal. The sarcoplasmic reticulum may have developed a unique retention mechanism(s) for these muscle-specific proteins.     

Drosophila signal peptide peptidase is an essential protease for larval development

We identified the Drosophila melanogaster Signal peptide peptidase gene (Spp) that encodes a multipass transmembrane aspartyl protease. Drosophila SPP is homologous to the human signal peptide peptidase (SPP) and is distantly related to the presenilins. We show that, like human SPP, Drosophila SPP can proteolyze a model signal peptide and is sensitive to an SPP protease inhibitor and that it localizes to the endoplasmic reticulum. Expression of Drosophila SPP was first apparent at germ band extension, and in late embryos it was robust in the salivary glands, proventriculus, and tracheae. Flies bearing mutations in conserved residues or carrying deficiencies for the Spp gene had defective tracheae and died as larvae.     

An Artificial Mitochondrial Tail Signal/Anchor Sequence Confirms a Requirement for Moderate Hydrophobicity for Targeting

Tail-anchored proteins are a group of membrane proteins oriented with their amino terminus in the cytoplasm and their carboxy terminus embedded in intracellular membranes. This group includes the apoptosis-mediating proteins of the Bcl-2 family as well as the vesicle targeting proteins of the SNARE group, among others. A stretch of hydrophobic amino acids at the extreme carboxy terminus of these proteins serves both as a membrane anchor and as a targeting signal. Tail-anchored proteins are differentially targeted to either the endoplasmic reticulum or the mitochondrial outer membrane and the mechanism which accomplishes this selective targeting is poorly understood. Here we define important characteristics of the signal/anchor region which directs proteins to the mitochondrial outer membrane. We have created an artificial sequence consisting of a stretch of 16 leucines bounded by positively charged amino acids. Using this template we demonstrate that moderate hydrophobicity distinguishes the mitochondrial tail-anchor sequence from that of the endoplasmic reticulum tail-anchor sequence. A change as small as introduction of a single polar residue into a sequence that otherwise targets to the endoplasmic reticulum can substantially switch targeting to the mitochondrial outer membrane. Further we show that a mitochondrially targeted tail-anchor has a higher propensity for the formation of alpha-helical structure than a sequence directing tail-anchored proteins to the endoplasmic reticulum.     

Phosphorylation of hepatitis B virus precore and core proteins.

Hepatitis B virus precore and core proteins are related. The precore protein contains the entire sequence of the core protein plus an amino-terminal extension of 29 amino acids. The amino-terminal extension of the precore protein contains a signal sequence for the secretion of the precore protein. This signal sequence is removed after the translocation of the precore protein across the endoplasmic reticulum membrane to produce the precore protein derivative named P22. We demonstrate that both P22 and the core protein can be phosphorylated in cells. Microsomal fractionation and trypsin digestion experiments demonstrate that a fraction of phosphorylated P22 is located in the endoplasmic reticulum lumen. Phosphorylation of P22 likely occurs in the carboxy terminus, since the P22 derivative P16, which lacks the carboxy terminus of P22, is not phosphorylated. Linking the carboxy terminus of the precore-core protein to heterologous secretory and cytosolic proteins led to the phosphorylation of the resulting chimeric proteins. These results indicate that phosphorylation of P22 and the core protein is likely mediated by cellular kinases.     

The type II isoform of bovine brain protein L-isoaspartyl methyltransferase has an endoplasmic reticulum retention signal (...RDEL) at its C-terminus.

Bovine brain is known to contain two major isoforms of protein L-isoaspartyl methyltransferase (PIMT), an enzyme that facilitates repair of atypical L-isoaspartyl peptide bonds in proteins. Although the two isoforms can be separated by anion-exchange chromatography, they appear to have similar, if not identical, substrate specificities in vitro. The more basic type I isoform has been extensively characterized, and its complete sequence has been reported. The present study was undertaken in an attempt to understand the structural and functional uniqueness of the more acidic type II isoform. Electrospray mass spectrometry of the intact enzymes revealed that the type II isoform is approximately 43 amu heavier than the type I isoform. Cyanogen bromide cleavage followed by HPLC with on-line mass analysis revealed that the type II isoform contains a unique C-terminal fragment which is 43 amu heavier than the corresponding fragment from the type I isoform. Amino acid composition analysis and direct sequencing of this fragment indicate that the type II isoform ends in the sequence ...RDEL, while the type I is known to end in ...RWK. Since ...RDEL, like ...KDEL, serves as an effective endoplasmic reticulum retention signal, we propose that the type II isoform serves to repair damaged proteins within the endoplasmic reticulum or, perhaps, within some other specialized compartment of the cell. Comparison of the protein sequences of the two bovine brain isoforms to DNA sequences for rodent PIMT reported by others suggests that the type II isoform may be produced by splicing within the codon for Arg224.     

Improved Production of the Human Granulocyte-Macrophage Colony Stimulating Factor in Transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Seeds Using a Dual Sorting Signal Peptide

The human granulocyte-macrophage colony stimulating factor (hGM-CSF) containing either an endoplasmic reticulum (ER) retention signal or a phaseolin vacuolar sorting signal peptide was expressed in Arabidopsis thaliana under the control of a tissue-specific promoter, derived from the soybean α′ subunit of β-conglycinin. No significant differences in recombinant hGM-CSF (rhGM-CSF) accumulation were detected between transgenic plants carrying either one of the two signal peptides. Hybrid seed from crosses between single-copy transformants tailed with the ER retention signal tetrapeptide and single-copy transformed plants tagged with a phaseolin four carboxy-terminal residues showed gene additive effects. The highest expression level of rhGM-CSF was 0.05% of total soluble protein of immature siliques, indicating that the two signal peptides functioned independently in the protein-sorting pathway. Additionally, TF-1 cell proliferation data demonstrated that rhGM-CSF was biologically active.     

Determinant for endoplasmic reticulum retention in the luminal domain of the human cytomegalovirus US3 glycoprotein

US3 of human cytomegalovirus is an endoplasmic reticulum resident transmembrane glycoprotein that binds to major histocompatibility complex class I molecules and prevents their departure. The endoplasmic reticulum retention signal of the US3 protein is contained in the luminal domain of the protein. To define the endoplasmic reticulum retention sequence in more detail, we have generated a series of deletion and point mutants of the US3 protein. By analyzing the rate of intracellular transport and immunolocalization of the mutants, we have identified Ser58, Glu63, and Lys64 as crucial for retention, suggesting that the retention signal of the US3 protein has a complex spatial arrangement and does not comprise a contiguous sequence of amino acids. We also show that a modified US3 protein with a mutation in any of these amino acids maintains its ability to bind class I molecules; however, such mutated proteins are no longer retained in the endoplasmic reticulum and are not able to block the cell surface expression of class I molecules. These findings indicate that the properties that allow the US3 glycoprotein to be localized in the endoplasmic reticulum and bind major histocompatibility complex class I molecules are located in different parts of the molecule and that the ability of US3 to block antigen presentation is due solely to its ability to retain class I molecules in the endoplasmic reticulum.     

Molecular cloning and nucleotide sequence of cDNA of microsomal carboxyesterase E1 of rat liver

cDNA clones of the mRNA for rat liver carboxyesterase E1, one of the carboxyesterases exclusively located on the luminal side of microsomal vesicles, were isolated. Sequence analysis of 2 kbp long cDNA revealed the primary structure of carboxyesterase E1, which consisted of 549 amino acids (Mr 60, 171.71) and contained an extra peptide of 18 amino acids at the NH2-terminus of the mature enzyme. Comparison of the deduced primary structure and sequences of some proteolytic fragments of the purified enzyme indicated the multiplicity of the enzyme. The extra peptide at the NH2-terminal had features in common with the signal peptides of most secretory proteins. However, no polar amino acid residues existed before the hydrophobic core of the signal peptide. A new interpretation is proposed to explain how the signal peptide without the NH2-terminal polar residues works. A tetrapeptide (KDEL) which was shown to keep a few microsomal proteins in the lumen of the endoplasmic reticulum was not found in the primary structure of carboxyesterase E1, which suggested the existence of another mechanism for retention of proteins in the lumen of endoplasmic reticulum. Carboxyesterase E1 showed significant homology with the COOH-terminal portion of thyroglobulin.     

Differential localization and identification of a critical aspartate suggest non-redundant proteolytic functions of the presenilin homologues SPPL2b and SPPL3

Signal peptide peptidase (SPP) is an unusual aspartyl protease that mediates clearance of signal peptides by proteolysis within the endoplasmic reticulum (ER). Like presenilins, which provide the proteolytically active subunit of the gamma-secretase complex, SPP contains a critical GXGD motif in its C-terminal catalytic center. Although SPP is known to be an aspartyl protease of the GXGD type, several presenilin homologues/SPP-like proteins (PSHs/SPPL) of unknown function have been identified by data base searches. We now investigated the subcellular localization and a putative proteolytic activity of PSHs/SPPLs in cultured cells and in an in vivo model. We demonstrate that SPPL2b is targeted through the secretory pathway to endosomes/lysosomes, whereas SPP and SPPL3 are restricted to the ER. As suggested by the differential subcellular localization of SPPL2b compared with SPP and SPPL3, we found distinct phenotypes upon antisense gripNA-mediated knockdown in zebrafish. spp and sppl3 knockdowns in zebrafish result in cell death within the central nervous system, whereas reduction of sppl2b expression causes erythrocyte accumulation in an enlarged caudal vein. Moreover, expression of D/A mutations of the putative C-terminal active sites of spp, sppl2, and sppl3 produced phenocopies of the respective knockdown phenotypes. Thus, our data suggest that all investigated PSHs/SPPLs are members of the novel family of GXGD aspartyl proteases. Furthermore, SPPL2b is shown to be the first member of the SPP/PSH/SPPL family that is not located within the ER but in endosomal/lysosomal vesicles.     

Efficiency and diversity of protein localization by random signal sequences.

Three randomly derived sequences that can substitute for the signal peptide of Saccharomyces cerevisiae invertase were tested for the efficiency with which they can translocate invertase or beta-galactosidase into the endoplasmic reticulum. The rate of translocation, as measured by glycosylation, was estimated in pulse-chase experiments to be less than 6 min. When fused to beta-galactosidase, these peptides, like the normal invertase signal sequence, direct the hybrid protein to a perinuclear region, consistent with localization to the endoplasmic reticulum. The diversity of function of random peptides was studied further by immunofluorescence localization of proteins fused to 28 random sequences: 4 directed the hybrid to the endoplasmic reticulum, 3 directed it to the mitochondria, and 1 directed it to the nucleus.     

The carboxylesterase family exhibits C-terminal sequence diversity reflecting the presence or absence of endoplasmic-reticulum-retention sequences.

Resident proteins of the endoplasmic reticulum lumen are continuously retrieved from an early Golgi compartment by a receptor-mediated mechanism. The sorting or retention sequence on the endoplasmic reticulum proteins is located at the C-terminus and was initially shown to be the tetrapeptide KDEL in mammalian cells and HDEL in Saccharomyces cerevisiae. The carboxylesterases are a large family of enzymes primarily localized to the lumen of the endoplasmic reticulum. Retention sequences in these proteins have been difficult to identify due to atypical and heterogeneous C-terminal sequences. Utilizing the polymerase chain reaction with degenerate primers, we have identified and characterized the C-termini of four members of the carboxylesterase family from rat liver. Three of the carboxylesterases sequences contained C-terminal sequences (HVEL, HNEL or HTEL) resembling the yeast sorting signal which were reported to be non-functional in mammalian cells. A fourth carboxylesterase contained a distinct C-terminal sequence, TEHT. A full-length esterase cDNA clone, terminating in the sequence HVEL, was isolated and was used to assess the retention capabilities of the various esterase C-terminal sequences. This esterase was retained in COS-1 cells, but was secreted when its C-terminal tetrapeptide, HVEL, was deleted. Addition of C-terminal sequences containing HNEL and HTEL resulted in efficient retention. However, the C-terminal sequence containing TEHT was not a functional retention signal. Both HDEL, the authentic yeast retention signal, and KDEL were efficient retention sequences for the esterase. These studies show that some members of the rat liver carboxylesterase family contain novel C-terminal retention sequences that resemble the yeast signal. At least one member of the family does not contain a C-terminal retention signal and probably represents a secretory form.     

ERGIC-53 KKAA signal mediates endoplasmic reticulum retrieval in yeast

Studies on the ERGIC-53 KKAA signal have revealed a new mechanism for static retention of mammalian proteins in the endoplasmic reticulum (Andersson, H., Kappeler, F., Hauri, H. P. (1999): Protein targeting to endoplasmic reticulum by dilysine signals involves direct retention in addition to retrieval. J. Biol. Chem. 274,15080 - 15084). To test if this mechanism was conserved in yeast, the ERGIC-53 KKAA signal was transferred on two different yeast reporter proteins. Making use of a genetic assay, we demonstrate that this signal induces COPI-dependent ER retrieval. ER retention of KKAA-tagged proteins was impaired in yeast mutants affected in COPI subunits. Furthermore, biochemical analysis of post-ER carbohydrate modifications detected on reporter proteins indicated that KKAA-tagged proteins recycle continuously within early compartments of the secretory pathway. Therefore in yeast, the KKAA signal might only function as a classical dilysine ER retrieval signal.     

Signal peptide peptidase-catalyzed cleavage of hepatitis C virus core protein is dispensable for virus budding but destabilizes the viral capsid

The capsid of hepatitis C virus (HCV) particles is considered to be composed of the mature form (p21) of core protein. Maturation to p21 involves cleavage of the transmembrane domain of the precursor form (p23) of core protein by signal peptide peptidase (SPP), a cellular protease embedded in the endoplasmic reticulum membrane. Here we have addressed whether SPP-catalyzed maturation to p21 is a prerequisite for HCV particle morphogenesis in the endoplasmic reticulum. HCV structural proteins were expressed by using recombinant Semliki Forest virus replicon in mammalian cells or recombinant baculovirus in insect cells, because these systems have been shown to allow the visualization of HCV budding events and the isolation of HCV-like particles, respectively. Inhibition of SPP-catalyzed cleavage of core protein by either an SPP inhibitor or HCV core mutations not only did not prevent but instead tended to facilitate the observation of viral buds and the recovery of virus-like particles. Remarkably, although maturation to p21 was only partially inhibited by mutations in insect cells, p23 was the only form of core protein found in HCV-like particles. Finally, newly developed assays demonstrated that p23 capsids are more stable than p21 capsids. These results show that SPP-catalyzed cleavage of core protein is dispensable for HCV budding but decreases the stability of the viral capsid. We propose a model in which p23 is the form of HCV core protein committed to virus assembly, and cleavage by SPP occurs during and/or after virus budding to predispose the capsid to subsequent disassembly in a new cell.     

The signal peptide of the G protein-coupled human endothelin B receptor is necessary for translocation of the N-terminal tail across the endoplasmic reticulum membrane

The initial step of the intracellular transport of G protein-coupled receptors, their insertion into the membrane of the endoplasmic reticulum, follows one of two different pathways. Whereas one group uses the first transmembrane domain of the mature receptor as an uncleaved signal anchor sequence for this process, a second group possesses additional cleavable signal peptides. The reason this second subset requires the additional signal peptide is not known. Here we have assessed the functional significance of the signal peptide of the endothelin B (ET(B)) receptor in transiently transfected COS.M6 cells. A green fluorescent protein-tagged ET(B) receptor mutant lacking the signal peptide was nonfunctional and retained in the endoplasmic reticulum, suggesting that it has a folding defect. To determine the defect in more detail, ET(B) receptor fragments containing the N-terminal tail, first transmembrane domain, and first cytoplasmic loop were constructed. We assessed N tail translocation across the endoplasmic reticulum membrane in the presence and absence of a signal peptide and show that the signal peptide is necessary for N tail translocation. We postulate that signal peptides are necessary for those G protein-coupled receptors for which post-translational translocation of the N terminus is impaired or blocked by the presence of stably folded domains.