Overexpression of an Arabidopsis gene encoding a subtilase (AtSBT5.4) produces a clavata-like phenotype

Arabidopsis thaliana encodes 56 subtilisin-like serine proteases (subtilases), and some are involved in the proteolytic processing of plant peptide hormones. Here, we have investigated the role of one subtilase, AtSBT5.4, in whole plant physiology by examining gain- or loss-of-function phenotypes. Knockouts of AtSBT5.4 had no apparent phenotype; however, overexpression produced a clavata-like phenotype with fasciated inflorescence stems and compounded terminal buds. Production of the phenotype depended on the enzymatic activity of the subtilase, because substitution of serine at the active site abolished the overexpression phenotype. When AtSBT5.4 was overexpressed in a clavata3 mutant background, a novel phenotype was produced suggesting that AtSBT5.4 interacts with the clavata signaling pathway. However, AtSBT5.4 did not cleave CLAVATA3 (CLV3) or a fluorogenic peptide representing the putative cleavage site in CLV3 under in vitro conditions suggesting that the interaction in vivo does not involve the cleavage of CLV3. Overexpression of AtSBT5.4 in a wuschel (wus) background suppressed the AtSBT5.4 overexpression phenotype indicating that WUS function is required for the AtSBT5.4 overexpression phenotype. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Diversity of Arabidopsis Genes Encoding Precursors for Phytosulfokine, a Peptide Growth Factor

Phytosulfokine-alpha (PSK-alpha), a unique plant peptide growth factor, was originally isolated from conditioned medium of asparagus (Asparagus officinalis) mesophyll cell cultures. PSK-alpha has several biological activities including promoting plant cell proliferation. Four genes that encode precursors of PSK-alpha have been identified from Arabidopsis. Analysis of cDNAs for two of these, AtPSK2 and AtPSK3, shows that both of these genes consist of two exons and one intron. The predicted precursors have N-terminal signal peptides and only a single PSK-alpha sequence located close to their carboxyl termini. Both precursors contain dibasic processing sites flanking PSK, analogous to animal and yeast prohormones. Although the PSK domain including the sequence of PSK-alpha and three amino acids preceding it are perfectly conserved, the precursors bear very limited similarity among Arabidopsis and rice (Oryza sativa), suggesting a new level of diversity among polypeptides that are processed into the same signaling molecule in plants, a scenario not found in animals and yeast. Unnatural [serine-4]PSK-beta was found to be secreted by transgenic Arabidopsis cells expressing a mutant of either AtPSK2 or AtPSK3 cDNAs, suggesting that both AtPSK2 and AtPSK3 encode PSK-alpha precursors. AtPSK2 and AtPSK3 were expressed demonstrably not only in cultured cells but also in intact plants, suggesting that PSK-alpha may be essential for plant cell proliferation in vivo as well as in vitro. Overexpression of either precursor gene allowed the transgenic calli to grow twice as large as the controls. However, the transgenic cells expressing either antisense cDNA did not dramatically decrease mitogenic activity, suggesting that these two genes may act redundantly.     

Cleavage of tRNA precursors by the RNA subunit of E. coli ribonuclease P (M1 RNA) is influenced by 3''-proximal CCA in the substrates

tRNA precursor molecules that contain the CCA sequence found at the 3' termini of all mature tRNAs are cleaved in vitro more readily by M1 RNA, the catalytic subunit of E. coli RNAase P, than precursors that lack this sequence. The sensitivity to the CCA sequence is not apparent when precursors are cleaved by the reconstituted RNAase P holoenzyme that contains both M1 RNA and the protein subunit. These results have been obtained with monomeric precursor molecules encoded by the E. coli and human chromosomes and with three dimeric precursor molecules encoded by the bacteriophage T4 genome. The data are in agreement with previous results concerning T4 tRNA biosynthesis in vivo and show that the CCA sequence is important for the processing of precursors to tRNAs.     

Dimer structure of a neuropeptide precursor established: consequences for processing

A prohormone (P1) of locust adipokinetic hormone I (AKH I) is shown here to be a homodimer of a 41 residue subunit called the A-chain. The A-chain, from the N terminal, consists of AKH I (10 amino acids starting with pyroglutamate) followed by a Gly-Lys-Arg processing site and then a 28 residues called the alpha chain containing a single cysteine and a potential Arg-Lys processing site. When processed each molecule of the homodimer precursor yields two copies of AKH I and one alpha chain homodimer. We call the alpha-alpha homodimer product of P1 processing AKH precursor related peptide 1 or APRP 1. The Arg-Lys dibasic pair found within the alpha chain is not cleaved in vivo. Our results show that neuropeptide precursors can be dimers and that dimer products can be synthesized by processing of a preformed dimer precursor rather than by dimerization of independent subunits.     

Identification of Arabidopsis subtilisin-like serine protease specifically expressed in root stele by gene trapping

Xylem plays a role not only in the transport of water and nutrients but also in the regulation of growth and development through the transport of biologically active substances. In addition to mineral salts, xylem sap contains hormones, organic nutrients and proteins. However, the physiological functions of most of those substances remain unclear. To explore genes involved in xylem sap production, we identified Arabidopsis genes expressed in the root stele of the root hair zone from gene-trap lines by randomly inserting the β-glucuronidase gene into the genome. Among 26 000 gene-trap lines, we found that 10 lines had β-glucuronidase (GUS) staining predominantly in the root stele of the root hair zone and no GUS staining in the shoots. Of these 10 lines, 2 lines showed that gene-trap tags inserted into the promoter region of the same gene, denoted Arabidopsis thaliana subtilase 4.12( AtSBT4.12 ). Analysis of AtSBT4.12 promoter using an pAtSBT4.12 ::β-glucuronidase transgenic line showed that the AtSBT4.12 gene was expressed only in the root stele of the root hair zone. AtSBT4.12 expression in roots was increased by application of methyl jasmonate. Subtilase proteins are commonly detected in proteomic analyses of xylem sap from various plant species, including Brassica napus , a relative of Arabidopsis . These results suggest that AtSBT4.12 may be a protein localized in the apoplast of root stele including xylem vessel and involved in stress responses in Arabidopsis roots.     

拟南芥植物硫肽激素-α前体基因AtPSK2的克隆及序列分析

根据拟南芥基因组数据库提供的信息,首次通过聚合酶链反应技术克隆到一个拟南芥硫肽激素-α前体基因——AtPSK2,并对其进行了序列分析。结果表明,所获得的AtPSK2基因是一个长412bp,含有一个内含子和两个没有3’或5’-非转译区的外显子的全编码序列,与数据库提供的序列比较,具有100％的同源性。这一工作将为转基因植物及其细胞培养和育种打下基础．     

Determinants for cleavage of the chlorophyll a/b binding protein precursor: a requirement for a basic residue that is not universal for chloroplast imported proteins

We demonstrate that the precursor of the major light-harvesting chlorophyll a/b binding protein (LHCP of Photosystem II), encoded by a Type I gene, contains distinct determinants for processing at two sites during in vitro import into the chloroplast. Using precursors from both pea and wheat, it is shown that primary site processing, and release of a approximately 26-kD peptide, depends on an amino-proximal basic residue. Substitution of an arginine at position -4 resulted in an 80% reduction in processing, with the concomitant accumulation of a high molecular weight intermediate. Cleavage occurred normally when arginine was changed to lysine. The hypothesis that a basic residue is a general requirement for transit peptide removal was tested. We find that the precursors for the small subunit of Rubisco and Rubisco activase do not require a basic residue within seven amino acids of the cleavage site for maturation. In the wheat LHCP precursor, determinants for efficient cleavage at a secondary site were identified carboxy to the primary site, beyond what is traditionally called the transit peptide, within the sequence ala-lys-ala-lys (residues 38-41). Introduction of this sequence into the pea precursor, which has the residues thr-thr-lys-lys in the corresponding position, converted it to a substrate with an efficiently recognized secondary site. Our results indicate that two different forms of LHCP can be produced with distinct NH2-termini by selective cleavage of a single precursor polypeptide.     

The isolation and characterization of rabbit motilin precursor cDNA.

The cDNA sequence of rabbit motilin precursor has been determined. The predicted amino acid sequence indicates that the precursor consists of 133 amino acids and includes a 25 amino acid signal peptide followed by the 22 amino acid motilin sequence and an 86 amino acid motilin associated peptide (MAP). As in the human and porcine precursors, two lysine residues follow motilin in the rabbit sequence. Rabbit motilin shares 64% amino acid sequence identity with human and porcine motilin, and all amino acid substitutions represent conservative changes. Amino acid sequence alignments of the rabbit, human and porcine MAP sequences suggest three functional/structural motifs corresponding to a putative endoproteinase recognition site, a putative PEST site and a potential posttranslational processing recognition element.     

Crimean-Congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase SKI-1

Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne member of the genus Nairovirus, family Bunyaviridae. The mature virus glycoproteins, Gn and Gc (previously referred to as G2 and G1), are generated by proteolytic cleavage from precursor proteins. The amino termini of Gn and Gc are immediately preceded by tetrapeptides RRLL and RKPL, respectively, leading to the hypothesis that SKI-1 or related proteases may be involved (A. J. Sanchez, M. J. Vincent, and S. T. Nichol, J. Virol. 76:7263-7275, 2002). In vitro peptide cleavage data show that an RRLL peptide representing the Gn processing site is efficiently cleaved by SKI-1 protease, whereas an RKPL peptide representing the Gc processing site is cleaved at negligible levels. The efficient cleavage of RRLL peptide is consistent with the known recognition sequences of SKI-1, including the sequence determinants involved in the cleavage of the Lassa virus (family Arenaviridae) glycoprotein precursor. These in vitro findings were confirmed by expression of wild-type or mutant CCHF virus glycoproteins in CHO cells engineered to express functional or nonfunctional SKI-1. Gn processing was found to be dependent on functional SKI-1, whereas Gc processing was not. Gn processing occurred in the endoplasmic reticulum-cis Golgi compartments and was dependent on an R at the -4 position within the RRLL recognition motif, consistent with the known cleavage properties of SKI-1. Comparison of SKI-1 cleavage efficiency between peptides representing Lassa virus GP2 and CCHF virus Gn cleavage sites suggests that amino acids flanking the RRLL may modulate the efficiency. The apparent lack of SKI-1 cleavage at the CCHF virus Gc RKPL site indicates that related proteases, other than SKI-1, are likely to be involved in the processing at this site and identical or similar sites utilized in several New World arenaviruses.     

Androgens affect the processing of secretory protein precursors in the guinea pig seminal vesicle. II. Identification of conserved sites for protein processing

The guinea pig seminal vesicle epithelium is an androgen-dependent tissue that synthesizes and secretes four major secretory proteins (SVP-1, SVP-2, SVP-3, and SVP-4). Sequencing of near full-length cDNA clones corresponding to the two most abundant mRNAs produced by the seminal vesicle reveals that all four secretory proteins are cleaved from two secretory protein precursors. Amino acid sequences from purified SVP-2 match the central region of the predicted amino acid sequences from the smaller cDNA clone, GP2 (581 nucleotides). Similar analysis demonstrates that the predicted amino acid sequence from the longer cDNA clone, GP1 (1368 nucleotides), codes for the related proteins SVP-3 and SVP-4 as well as SVP-1. The 43.2 kilodalton polyprotein precursor coded by GP1 contains two different sets of 24 amino acid tandemly repeated sequences. The two secretory protein precursors have extensive regions of peptide sequence homology, particularly in regions where protein processing must occur to produce the mature secretory proteins. Analysis of the predicted secondary structure of the two precursor polypeptides revealed a strong correlation between structural features and sites of protein processing.     

Import into chloroplasts of a yeast mitochondrial protein directed by ferredoxin and plastocyanin transit peptides

Many chloroplast proteins are synthesized in the cytoplasm as precursors which contain an amino terminal transit peptide. These precursors are subsequently imported into chloroplast and targeted to one of several organellar locations. This import is mediated by the transit peptide, which is cleaved off during import. We have used the transit peptides of ferredoxin (chloroplast stroma) and plastocyanin (thylakoid lumen) to study chloroplast protein import and intra-organellar routing toward different compartments. Chimeric genes were constructed that encode precursor proteins in which the transit peptides are linked to yeast mitochondrial manganese superoxide dismutase. Chloroplast protein import and localization experiments show that both chimeric proteins are imported into the chloroplast stroma and processed. The plastocyanin transit sequence did not direct superoxide dismutase to the thylakoids; this protein was found in the stroma as an intermediate that still contains part of the plastocyanin transit peptide. The organelle specificity of these chimeric precursors reflected the transit peptide parts of the molecules, because neither the ferredoxin and plastocyanin precursors nor the chimeric proteins were imported into isolated yeast mitochondria.     

Thylakoid DeltapH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport

The thylakoid DeltapH-dependent pathway transports folded proteins with twin arginine-containing signal peptides. Identified components of the machinery include cpTatC, Hcf106, and Tha4. The reaction occurs in two steps: precursor binding to the machinery, and transport across the membrane. Here, we show that a cpTatC-Hcf106 complex serves as receptor for specific binding of twin arginine-containing precursors. Antibodies to either Hcf106 or cpTatC, but not Tha4, inhibited precursor binding. Blue native gel electrophoresis and coimmunoprecipitation of digitonin-solubilized thylakoids showed that Hcf106 and cpTatC are members of an approximately 700-kD complex that lacks Tha4. Thylakoid-bound precursor proteins were also associated with an approximately 700-kD complex and were coimmunoprecipitated with antibodies to cpTatC or Hcf106. Chemical cross-linking revealed that precursors make direct contact with cpTatC and Hcf106 and confirmed that Tha4 is not associated with precursor, cpTatC, or Hcf106 in the membrane. Precursor binding to the cpTatC-Hcf106 complex required both the twin arginine and the hydrophobic core of the signal peptide. Precursors remained bound to the complex when Tha4 was sequestered by antibody, even in the presence of DeltapH. These results indicate that precursor binding to the cpTatC-Hcf106 complex constitutes the recognition event for this pathway and that subsequent participation by Tha4 leads to translocation.     

Role for the P4 amino acid residue in substrate utilization by the poliovirus 3CD proteinase.

Amino acid insertions or substitutions were introduced into the poliovirus P1 capsid precursor at locations proximal to the two known Q-G cleavage sites to examine the role of the P4 residue in substrate processing by proteinase 3CD. Analysis of the processing profile of P1 precursors containing four-amino-acid insertions into the carboxy terminus of VP3 or a single-amino-acid substitution at the P4 position of the VP3-VP1 cleavage site demonstrates that substitution of the alanine residue in the P4 position of the VP3-VP1 cleavage site significantly affects cleavage at that site by proteinase 3CD. A single-amino-acid substitution at the P4 position of the VP0-VP3 cleavage site, on the other hand, has only a slight effect on 3CD-mediated processing at this cleavage site. Finally, analysis of six amino acid insertion mutations containing Q-G amino acid pairs demonstrates that the in vitro and in vivo selection of a cleavage site from two adjacent Q-G amino acid pairs depends on the presence of an alanine in the P4 position of the cleaved site. Our data provide genetic and biochemical evidence that the alanine residue in the P4 position of the VP3-VP1 cleavage site is a required substrate determinant for the recognition and cleavage of that site by proteinase 3CD and suggest that the P4 alanine residue may be specifically recognized by proteinase 3CD.     

Import of rat ornithine transcarbamylase precursor into mitochondria: two-step processing of the leader peptide

The mitochondrial matrix enzyme ornithine transcarbamylase (OTC) is synthesized on cytoplasmic polyribosomes as a precursor (pOTC) with an NH2-terminal extension of 32 amino acids. We report here that rat pOTC synthesized in vitro is internalized and cleaved by isolated rat liver mitochondria in two, temporally separate steps. In the first step, which is dependent upon an intact mitochondrial membrane potential, pOTC is translocated into mitochondria and cleaved by a matrix protease to a product designated iOTC, intermediate in size between pOTC and mature OTC. This product is in a trypsin-protected mitochondrial location. The same intermediate-sized OTC is produced in vivo in frog oocytes injected with in vitro-synthesized pOTC. The proteolytic processing of pOTC to iOTC involves the removal of 24 amino acids from the NH2 terminus of the precursor and utilizes a cleavage site two residues away from a critical arginine residue at position 23. In a second cleavage step, also catalyzed by a matrix protease, iOTC is converted to mature OTC by removal of the remaining eight residues of leader sequence. To define the critical regions in the OTC leader peptide required for these events, we have synthesized OTC precursors with alterations in the leader. Substitution of either an acidic (aspartate) or a "helix-breaking" (glycine) amino acid residue for arginine 23 of the leader inhibits formation of both iOTC and OTC, without affecting translocation. These mutant precursors are cleaved at an otherwise cryptic cleavage site between residues 16 and 17 of the leader. Interestingly, this cleavage occurs at a site two residues away from an arginine at position 15. The data indicate that conversion of pOTC to mature OTC proceeds via the formation of a third discrete species: an intermediate-sized OTC. The data suggest further that, in the rat pOTC leader, the essential elements required for translocation differ from those necessary for correct cleavage to either iOTC or mature OTC.     

Mono- and dibasic proteolytic cleavage sites in insect neuroendocrine peptide precursors

Regulatory peptides are synthesized as part of larger precursors that are subsequently processed into the active substances. After cleavage of the signal peptide, further proteolytic processing occurs predominantly at basic amino acid residues. Rules have been proposed in order to predict which putative proteolytic processing sites are actually used, but these rules have been established for vertebrate peptide precursors and it is unclear whether they are also valid for insects. The aim of this paper is to establish the validity of these rules to predict proteolytic cleavage sites at basic amino acids in insect neuropeptide precursors. Rules describing the cleavage of mono- and dibasic potential processing sites in insect neuropeptide precursors are summarized below. Lys-Arg pairs not followed by an aliphatic or basic amino acid residue are virtually always cleaved in insect regulatory peptide precursors, but cleavages of Lys-Arg pairs followed by either an aliphatic or a basic amino acid residue are ambiguous, as is processing at Arg-Arg pairs. Processing at Arg-Lys pairs has so far not been demonstrated in insects and processing at Lys-Lys pairs appears very rare. Processing at single Arg residues occurs only when there is a basic amino acid residue in position -4, -6, or -8, usually an Arg, but Lys or His residues work also. Although the current number of such sites is too limited to draw definitive conclusions, it seems plausible that cleavage at these sites is inhibited by the presence of aliphatic residues in the +1 position. However, cleavage at single Arg residues is ambiguous. When several potential cleavage sites overlap the one most easily cleaved appears to be processed. It cannot be excluded that some of the rules formulated here will prove less than universal, as only a limited number of cleavage sites have so far been identified. It is likely that, as in vertebrates, ambiguous processing sites exist to allow differential cleavage of the same precursor by different convertases and it seems possible that the precursors of allatostatins and PBAN are differentially cleaved in different cell types. Arch. Insect Biochem. Physiol. 43:49-63, 2000.     

Proteolytic activity of novel human immunodeficiency virus type 1 proteinase proteins from a precursor with a blocking mutation at the N terminus of the PR domain.

The mature human immunodeficiency virus type 1 proteinase (PR; 11 kDa) can cleave all interdomain junctions in the Gag and Gag-Pol polyprotein precursors. To determine the activity of the enzyme in its precursor form, we blocked release of mature PR from a truncated Gag-Pol polyprotein by introducing mutations into the N-terminal Phe-Pro cleavage site of the PR domain. The mutant precursor autoprocessed efficiently upon expression in Escherichia coli. No detectable mature PR was released; however, several PR-related products ranging in size from approximately 14 to 18 kDa accumulated. Products of the same size were generated when mutant precursors were digested with wild-type PR. Thus, PR can utilize cleavage sites in the region upstream of the PR domain, resulting in the formation of extended PR species. On the basis of active-site titration, the PR species generated from mutated precursor exhibited wild-type activity on peptide substrates. However, the proteolytic activity of these extended enzymes on polyprotein substrates provided exogenously was low when equimolar amounts of extended and wild-type PR proteins were compared. Mammalian cells expressing the mutated precursor produced predominantly precursor and considerably reduced amounts of mature products. Released particles consisted mostly of uncleaved or partially cleaved polyproteins. Our results suggest that precursor forms of PR can autoprocess but are less efficient in processing of the Gag precursor for formation of mature virus particles.     

Soluble Chloroplast Enzyme Cleaves preLHCP Made in Escherichia coli to a Mature Form Lacking a Basic N-Terminal Domain

We have investigated the specificity of a chloroplast soluble processing enzyme that cleaves the precursor of the major light-harvesting chlorophyll a/b binding protein (LHCP). The precursor of LHCP (preLHCP) was synthesized in Escherichia coli and recovered from inclusion-like bodies. It was found to be a substrate for proteolytic cleavage by the soluble enzyme in an organelle-free reaction, yielding a 25 kilodalton peptide. This peptide co-migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the smaller of the forms (25 and 26 kilodalton) produced when either the E. coli-synthesized precursor, or preLHCP made in a reticulocyte lysate, was imported into chloroplasts. N-Terminal sequence analysis of the E. coli-generated precursor showed that it lacked an N-terminal methionine. N-Terminal sequencing of the 25 kilodalton peptide produced in the organelle-free reaction indicated that processing occurred between residues 40 and 41, removing a basic domain (RKTAAK) thought to be at the N-terminus of all LHCP molecules of type I associated with photosystem II. To determine if the soluble enzyme involved also cleaves other precursor polypeptides, or is specific to preLHCP, it was partially purified, and the precursors for Rubisco small subunit, plastocyanin, Rubisco activase, heat shock protein 21, and acyl carrier protein were tested as substrates. All of these precursors were cleaved by the same chromatographic peak of activity that processes preLHCP in the organelle-free reaction.