Secretory Precursor Research Articles

SSS1 encodes a stabilizing component of the Sec61 subcomplex of the yeast protein translocation apparatus.

The yeast SSS1 gene has been isolated as an extragenic high copy suppressor of sec61, a mutant displaying defects in protein translocation into the endoplasmic reticulum (ER). We found that SSS1 is an essential gene required for transfer of secretory precursors through the ER membrane. Here we demonstrate that the SSS1 product (Sss1p) is firmly bound to the ER membrane and exposes its amino-terminal half on the cytosolic side. Only detergent, or an alkali treatment, is effective at extracting Sss1p from the membrane. Coimmunoprecipitation experiments revealed that Sss1p and Sec61p participate in the same multisubunit complex. Cross-linking followed by immunoprecipitation specifically yielded an additional polypeptide of molecular mass 73 kDa. Moreover, Sss1p and Sec61p show mutually stabilizing interactions: Sss1p is destabilized in a sec61 mutant context, and mutated Sec61p is stabilized by Sss1p overproduction. These observations account for the isolation of SSS1 as a dosage-dependent suppressor of sec61. Since the polytopic integral membrane protein Sec61p is adjacent to translocating precursors and to ribosomes, and given the comparable translocation deficiencies of sss1 or sec61 mutants, we propose that Sss1p belongs to the "Sec61 subcomplex" that constitutes the pore of the membrane-bound translocation apparatus.

Yeast beta- and beta'-coat proteins (COP). Two coatomer subunits essential for endoplasmic reticulum-to-Golgi protein traffic.

To understand better the role of non-clathrin coat proteins in membrane traffic, we have cloned and characterized two essential genes encoding subunits of the yeast coatomer, SEC26 and SEC27. Sec26p is a 109-kDa protein that shares 43% sequence identity with mammalian beta-coat protein (beta-COP). Sec26p-depleted cells accumulate endoplasmic reticulum (ER) forms of secretory precursor proteins, and growth ceases after a dramatic accumulation of ER membranes. Sec26p overproduction partially suppresses sec27-1, a new mutant that shows a temperature-sensitive defect in ER-to-Golgi transport. The SEC27 gene was cloned, and the sequence predicts a 99.4-kDa protein with 45% sequence identity to mammalian beta'-COP. Our sequence data support a two-domain model for the SEC27 protein: a conserved amino-terminal domain, composed of five WD-40 repeats similar to those found in beta-subunits of trimeric G proteins, and a less conserved carboxyl-terminal domain. Genetic interactions connect sec27-1 and sec21-1 (coatomer gamma subunit) with the ARF1 and ARF2 genes and with the SEC22, BET1, and BOS1 genes, which encode membrane proteins involved in ER-to-Golgi transport.

Sec72p contributes to the selective recognition of signal peptides by the secretory polypeptide translocation complex.

SEC72 encodes the 23-kD subunit of the Sec63p complex, an integral ER membrane protein complex that is required for translocation of presecretory proteins into the ER of Saccharomyces cerevisiae. DNA sequence analysis of SEC72 predicts a 21.6-kD protein with neither a signal peptide nor any transmembrane domains. Antibodies directed against a carboxyl-terminal peptide of Sec72p were used to confirm the membrane location of the protein. SEC72 is not essential for yeast cell growth, although an sec72 null mutant accumulates a subset of secretory precursors in vivo. Experiments using signal peptide chimeric proteins demonstrate that the sec72 translocation defect is associated with the signal peptide rather than with the mature region of the secretory precursor.

Apolipoprotein B48-membrane interactions. Absence of transmembrane localization in nonhepatic cells.

Apolipoprotein B (apoB) is essential for the hepatic assembly and secretion of triglyceride-rich very low density lipoproteins. Recent studies have revealed that in both hepatic and nonhepatic cells a large percentage of newly synthesized apoB polypeptides engage in transmembrane interactions with the endoplasmic reticulum (ER). These apoB-membrane interactions have been implicated in the processes of lipoprotein assembly and regulation. To identify domains of apoB that are responsible for its transmembrane localization, overlapping 300 amino acid segments of human apoB48 (amino-terminal 48% of apoB) and two control proteins, human complement component C3 and mouse CD4, were appended to the amino-terminal 77 amino acids of a soluble secretory precursor protein and expressed in COS-1 cells. While the integral membrane protein CD4 conferred predictable transmembrane orientation on the hybrid protein, as evidenced by its partial protease accessibility in intact microsomes, all of the apoB-containing proteins and the soluble secretory control, C3, were fully protease-resistant, consistent with their complete translocation into the ER. To determine if conformational properties of apoB are responsible for its transmembrane interactions with the ER, proteins containing the entire amino-terminal approximately 50% of apoB (apoB50) were expressed in COS-1 cells. Irrespective of whether targeting and translocation initiation were directed by a heterologous signal peptide or the native apoB signal peptide, apoB50 appeared to undergo complete membrane translocation into a protease-inaccessible compartment. These results demonstrate that the amino-terminal 50% of apoB lacks autonomous signals or properties that can fully block ER membrane translocation or promote any other form of stable transmembrane assembly in nonhepatic cells.

Structural and functional characterization of Sec66p, a new subunit of the polypeptide translocation apparatus in the yeast endoplasmic reticulum.

SEC66 encodes the 31.5-kDa glycoprotein of the Sec63p complex, an integral endoplasmic reticulum membrane protein complex required for translocation of presecretory proteins in Saccharomyces cerevisiae. DNA sequence analysis of SEC66 predicts a 23-kDa protein with no obvious NH2-terminal signal sequence but with one domain of sufficient length and hydrophobicity to span a lipid bilayer. Antibodies directed against a recombinant form of Sec66p were used to confirm the membrane location of Sec66p and that Sec66p is a glycoprotein of 31.5 kDa. A null mutation in SEC66 renders yeast cells temperature sensitive for growth. sec66 cells accumulate some secretory precursors at a permissive temperature and a variety of precursors at the restrictive temperature. sec66 cells show defects in Sec63p complex formation. Because sec66 cells affect the translocation of some, but not all secretory precursor polypeptides, the role of Sec66p may be to interact with the signal peptide of presecretory proteins.

Folding and intracellular transport of the yeast plasma-membrane H(+)-ATPase: effects of mutations in KAR2 and SEC65.

We have developed two independent assays to study the integration, folding, and intracellular transport of the polytopic plasma membrane H(+)-ATPase in yeast. To follow folding, controlled trypsinolysis was used to distinguish between the E1 conformation of the ATPase (favored in the presence of ADP) and the E2 conformation (favored in the presence of vanadate). By this criterion, wild-type ATPase appears to recognize its ligands and assume distinct conformations within a short time after its biosynthesis. To follow intracellular transport, we have exploited the fact that export of newly synthesized ATPase from the endoplasmic reticulum is accompanied by kinase-mediated phosphorylation, leading to a shift in electrophoretic mobility. Because proper folding is required for transport from the endoplasmic reticulum, the mobility shift also serves as a convenient bioassay for correct folding. As a first step toward identifying cell components important in folding of the nascent ATPase, we have used the dual assays to examine the role of KAR2, encoding the yeast homolog of immunoglobulin heavy chain binding protein/78-kDa glucose-regulated protein, and SEC65, encoding a subunit of the yeast signal recognition particle. Although mutation of KAR2 caused defective translocation of several secretory precursors into the endoplasmic reticulum lumen, ATPase folding and intracellular transport were unperturbed. By contrast, in a sec65 mutant, the folding and intracellular transport of newly synthesized ATPase were delayed. Our data suggest that conformational maturation of the ATPase is a rapid process in wild-type cells and that membrane integration mediated by signal recognition peptide is important for the proper folding of this polytopic protein.

The 70-Kilodalton Heat Shock Cognate Can Act as a Molecular Chaperone during the Membrane Translocation of a Plant Secretory Protein Precursor.

When a model secretory precursor was synthesized in vitro and analyzed by rate-zonal sedimentation, it appeared to be associated with other proteins present in a wheat germ extract. At least one of the associated proteins is a member of the 70-kD family of stress proteins. It was possible to immunoprecipitate the secretory precursor with anti-heat shock cognate 70 (Hsc70) antibodies in the absence but not in the presence of ATP, suggesting that the association was specific. ATP-sensitive association is one diagnostic characteristic of molecular chaperone-type proteins. Increasing incubation temperature decreased the amount of precursor associated with Hsc70. A method was developed for the removal of Hsc70 from a wheat germ in vitro translation mixture by immunoprecipitation. Cotranslational translocation and processing of the secretory precursor by maize endosperm microsomes were inefficient in the Hsc70-depleted system but were greatly stimulated by addition of purified preparations of various heat shock 70 proteins (Hsp70s). Cytosolic Hsc70 from maize endosperm was capable of autophosphorylation in vitro. Phosphorylated Hsc70 was much less efficient in promoting membrane translocation of the secretory precursor. These results suggest that chaperone function in vivo could be regulated by phosphorylation.

The 70-Kilodalton Heat Shock Cognate Can Act as a Molecular Chaperone during the Membrane Translocation of a Plant Secretory Protein Precursor

The 70-Kilodalton Heat Shock Cognate Can Act as a Molecular Chaperone during the Membrane Translocation of a Plant Secretory Protein Precursor

The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation.

Detergent extracts of canine pancreas rough microsomal membranes were depleted of either the signal recognition particle receptor (SR), which mediates the signal recognition particle (SRP)-dependent targeting of the ribosome/nascent chain complex to the membrane, or the signal sequence receptor (SSR), which has been proposed to function as a membrane bound receptor for the newly targeted nascent chain and/or as a component of a multi-protein translocation complex responsible for transfer of the nascent chain across the membrane. Depletion of the two components was performed by chromatography of detergent extracts on immunoaffinity supports. Detergent extracts lacking either SR or SSR were reconstituted and assayed for activity with respect to SR dependent elongation arrest release, nascent chain targeting, ribosome binding, secretory precursor translocation, and membrane protein integration. Depletion of SR resulted in the loss of elongation arrest release activity, nascent chain targeting, secretory protein translocation, and membrane protein integration, although ribosome binding was unaffected. Full activity was restored by addition of immunoaffinity purified SR before reconstitution of the detergent extract. Surprisingly, depletion of SSR was without effect on any of the assayed activities, indicating that SSR is either not required for translocation or is one of a family of functionally redundant components.

Synthesis of precursor maltose-binding protein with proline in the +1 position of the cleavage site interferes with the activity of Escherichia coli signal peptidase I in vivo.

The residues occupying the -3 and -1 positions relative to the cleavage site of secretory precursor proteins are usually amino acids with small, neutral side chains that are thought to constitute the recognition site for the processing enzyme, signal peptidase. No restrictions have been established for residues positioned +1 to the cleavage site, although there have been several indications that mutant precursor proteins with a proline at +1 cannot be processed by Escherichia coli signal peptidase I (also called leader peptidase). A maltose-binding protein (MBP) species with proline at +1, designated MBP27-P, was translocated efficiently but not processed when expressed in E. coli cells. Unexpectedly, induced expression of MBP27-P was found to have an adverse effect on the processing kinetics of five different nonlipoprotein precursors analyzed, but not precursor Lpp (the major outer membrane lipoprotein) processed by a different enzyme, signal peptidase II. Cell growth also was inhibited following induction of MBP27-P synthesis. Substitutions in the MBP27-P signal peptide that blocked MBP translocation across the cytoplasmic membrane and, hence, access to the processing enzyme or that altered the signal peptidase I recognition site at position -1 restored both normal growth and processing of other precursors. Since overproduction of signal peptidase I also restored normal growth and processing to cells expressing unaltered MBP27-P, it was concluded that precursor MBP27-P interferes with the activity of the processing enzyme, probably by competing as a noncleavable substrate for the enzyme's active site. Thus, although signal peptidase I, like many other proteases, is unable to cleave an X-Pro bond, a proline at +1 does not prevent the enzyme from recognizing the normal processing site. When the RBP signal peptide was substituted for the MBP signal peptide of MBP27-P, the resultant hybrid protein was processed somewhat inefficiently at an alternate cleavage site and elicited a much reduced effect on cell growth and signal peptidase I activity. Although the MBP signal peptide also has an alternate cleavage site, the different properties of the RBP and MBP signal peptides with regard to the substitution of proline at +1 may be related to their respective secondary structures in the processing site region.

マルトペンタオース生成酵素の生産とその応用

The gene coding for the maltopentaose (G5)-forming enzyme of Pseudonaonas sp. KO-8940 was cloned into Escherichia coli and its nucleotides sequenced. It was found to have a long open reading frame composed of 1842 by that encoded 614 amino acid residues for a secretory precursor polypeptide including the typical signal sequence with an NH2-terminal. In the deduced primary structure of this enzyme, a high degree of homology to four regions conserved by many α-amylases was found, and the COOH-terminal portion of this enzyme showed high homology with other raw starch digesting amylases. The G5-forming enzyme was produced in large amount (52.7 IU/ml, 0.1 g/l) in E. coli under the tac promoter. This result showed that the G5-forming enzyme can be produced in E. coli carrying this enzyme gene expression vector at levels up to 6 times greater than the native production system found in P, sp. KO-8940.

Non-functional expression of Escherichia coli signal peptidase I in Bacillus subtilis

The Escherichia coli lep gene, encoding signal peptidase I (SPase I) was provided with Bacillus subtilis transcription/translation signals and expressed in this organism. When present on a low-copy-number plasmid, the amount of E. coli SPase I produced (per mg cell protein) in B. subtilis was half that produced in wild-type E. coli cells. The production of E. coli SPase I in B. subtilis was increased approximately fivefold by cloning the lep gene into a high-copy-number plasmid. The expression of E. coli SPase I in B. subtilis did not appear to increase the rate of processing of two hybrid secretory precursor proteins. Two observations may explain the failure of E. coli SPase I to stimulate processing of exported proteins in B. subtilis. First, the E. coli SPase I was apparently not exposed on the outside of the B. subtilis cytoplasmic membrane, indicating its incorrect insertion into the membrane. Second, in vitro processing studies, using cell-free extracts of B. subtilis producing E. coli SPase I, suggested that the enzyme was not active. A further outcome of this study was that conditions favouring processing of precursors by SPase I in cell-free extracts of E. coli did not favour processing by the corresponding enzyme in B. subtilis cell-free extracts. This suggests that significant differences exist between the two enzymes. The observation that antibodies directed against E. coli SPase I did not cross-react with B. subtilis membrane proteins supports this idea.

Protein U, a late-developmental spore coat protein of Myxococcus xanthus, is a secretory protein

Protein U is a spore coat protein produced at the late stage of development of Myxococcus xanthus. This protein was isolated from developmental cells, and its amino-terminal sequence was determined. On the basis of this sequence, the gene for protein U (pru) was cloned and its DNA sequence was determined, revealing an open reading frame of 179 codons. The product from this open reading frame has a typical signal peptide of 25 amino acid residues at the amino terminal end, followed by protein U of 154 residues. This result indicates that protein U is produced as a secretory precursor, pro-protein U, which is then secreted across the membrane to assemble on the spore surface. This is in sharp contrast to protein S, a major spore coat protein produced early in development, which has no signal peptide, indicating that there are two distinct pathways for trafficking of spore coat proteins during the differentiation of M. xanthus.

Alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases

The nucleotide sequence of the alpha-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the alpha-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature alpha-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 alpha-amylase with those from other bacterial and eucaryotic alpha-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca(2+)-binding site (consensus region I) of this Ca(2+)-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the alpha-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the beta-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.

Characterization of different forms of yeast acid trehalase in the secretory pathway

The biosynthesis and processing of the vacuolar (lysosomal) acid trehalase (molecular mass about 220 kDa) was followed in vivo using mutants conditionally defective in the secretory pathway. A precursor of 41 kDa was found in sec61 mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum. Endoglycosidase H and N-glycosidase F treatment of purified acid trehalase in vitro resulted in a 41 kDa band, indicating that the precursor form found in sec61 mutant cells corresponds to the carbohydrate-free form of the enzyme. sec18 mutant cells, blocked in the delivery of secretory proteins from the endoplasmic reticulum to the Golgi body accumulate a form with a molecular mass of 76 kDa which probably corresponds to a partially glycosylated precursor of the mature acid trehalase. This precursor partially disappears in favour of the appearance of a higher molecular weight component of 180 kDa in sec7 mutants which are blocked in the delivery step of secretory proteins from the Golgi body to the vacuole. In wild-type cells the fully glycosylated mature form of acid trehalase of about 220 kDa was observed accompanied by some 180 kDa and 76 kDa material.

Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast.

BiP/GRP78 is an essential member of the HSP70 family that resides in the lumen of the endoplasmic reticulum. In yeast, BiP/GRP78 is encoded by the KAR2 gene. A temperature sensitive mutation was isolated in KAR2 and found to cause a rapid block in protein secretion. Secretory precursors of a number of proteins (invertase, carboxypeptidase Y, alpha-factor, and BiP) accumulated that were characteristic of a block in translocation into the lumen of the ER. Protease protection experiments confirmed that the precursors accumulated on the cytoplasmic side of the ER membrane. Moreover, depletion of wild-type KAR2 protein also resulted in a block in translocation of secretory proteins. These results implicate BiP/GRP78 function in the continued translocation of proteins into the lumen of the ER.

SecA protein: Autoregulated initiator of secretory precursor protein translocation across theE. coli plasma membrane

Several classes of secA mutants have been isolated which reveal the essential role of this gene product for E. coli cell envelope protein secretion. SecA-dependent, in vitro protein translocation systems have been utilized to show that SecA is an essential, plasma membrane-associated, protein translocation factor, and that SecA's ATPase activity appears to play an essential but as yet undefined role in this process. Cell fractionation studies suggested that SecA protein is in a dynamic state within the cell, occurring in soluble, peripheral, and integral membraneous states. These data have been used to argue that SecA is likely to promote the initial insertion of secretory precursor proteins into the plasma membrane in a manner dependent on ATP hydrolysis. The protein secretion capability of the cell has been shown to translationally regulate secA expression with SecA protein serving as an autogenous repressor, although the exact mechanism and purpose of this regulation need to be defined further.

Receptor-mediated binding of secretory protein precursors to endoplasmic reticulum membranes in yeast

Receptor-mediated binding of secretory protein precursors to endoplasmic reticulum membranes in yeast

The role of the mature part of secretory proteins in translocation across the plasma membrane and in regulation of their synthesis in Escherichia coli

Presently available data are reviewed which concern the role of the mature parts of secretory precursor proteins in translocation across the plasma membrane of Escherichia coli. The following conclusions can be drawn; i) signals, acting in a positive fashion and required for translocation do not appear to exist in the mature polypeptides; ii) a number of features have been identified which either affect the efficiency of translocation or cause export incompatibility. These are: α) protein folding prior to translocation; β) restrictions regarding the structure of N-therminus; γ) presence of lipophilic anchors; δ) too low a size of the precursor. Efficiency of translocation is also enhanced by binding of chaperonins (SecB, trigger factor, GroEL) to precursors. Binding sites for chaperonins appear to exist within the mature parts of the precursors but the nature of these sites has remained rather mysterious. Mutant periplasmic proteins with a block in release from the plasma membrane have been described, the mechanism of this block is not known. The mature parts of secretory proteins can also be involved in the regulation of their synthesis. It appears that exported proteins are already recognized as such before they are channelled into the export pathway and that their synthesis can be feed-back inhibited at the translational level.

SEC62 encodes a putative membrane protein required for protein translocation into the yeast endoplasmic reticulum.

Yeast sec62 mutant cells are defective in the translocation of several secretory precursor proteins into the lumen of the endoplasmic reticulum (Rothblatt et al., 1989). The deficiency, which is most restrictive for alpha-factor precursor (pp alpha F) and preprocarboxypeptidase Y, has been reproduced in vitro. Membranes isolated from mutant cells display low and labile translocation activity with pp alpha F translated in a wild-type cytosol fraction. The defect is unique to the membrane fraction because cytosol from mutant cells supports translocation into membranes from wild-type yeast. Invertase assembly is only partly affected by the sec62 mutation in vivo and is nearly normal with mutant membranes in vitro. A potential membrane location for the SEC62 gene product is supported by evaluation of the molecular clone. DNA sequence analysis reveals a 32-kD protein with no obvious NH2-terminal signal sequence but with two domains of sufficient length and hydrophobicity to span a lipid bilayer. Sec62p is predicted to display significant NH2- and COOH-terminal hydrophilic domains on the cytoplasmic surface of the ER membrane. The last 30 amino acids of the COOH terminus may form an alpha-helix with 14 lysine and arginine residues arranged uniformly about the helix. This domain may allow Sec62p to interact with other proteins of the putative translocation complex.