A novel type of FKBP in the secretory pathway of Neurospora crassa

FKBPs define a subfamily of peptidyl-prolyl cis/trans isomerases (PPIases). PPIases are known to play roles in cellular protein folding, protein interactions and signal transduction. Here we describe NcFKBP22 from Neurospora crassa, a novel type of FKBP. NcFKBP22 is synthesized as a precursor protein with a cleavable signal sequence. In addition to a typical FKBP domain in the amino-terminal part mature NcFKBP22 contains a novel second domain which is unique amongst all known FKBPs. The amino acid composition of this carboxy-terminal domain is highly biased. Secondary structure predictions suggest that this domain may form an amphipathic -helix. The carboxy-terminus of NcFKBP22 is –HNEL, a potential endoplasmic reticulum (ER) retention signal, suggesting that NcFKBP22 is a resident protein of the ER.     

cDNA cloning of halocidin and a new antimicrobial peptide derived from the N-terminus of Ci-META4

Halocidin is an antimicrobial peptide, which is isolated from hemocytes from the tunicate, Halocynthia aurantium. In this study, we cloned the full-length cDNA of halocidin from pharyngeal tissue, using a combination of RT-PCR and 5′-RACE-PCR. The observed cDNA structure indicated that halocidin is synthesized as a 10.37 kDa prepropeptide. Based on the cDNA structure and the known amino acid sequence of the mature peptide, it was concluded that the precursor of halocidin contains a 21-residue signal peptide, followed by the 18 residues of the mature peptide, and a 56-residue anionic C-terminal extension, which is removed later on in the process. The signal sequence of halocidin exhibited a high degree of similarity with the corresponding portion of the Ci-META4 protein, which had been previously discovered in the coelomic cells of another tunicate, Ciona intestinalis, and is considered to play a role in metamorphosis. However, in several respects, the cDNA structure of Ci-META4 suggested that it might constitute a precursor for an antimicrobial peptide. Thus, we prepared a synthetic peptide, which was comprised of 19 N-terminal amino acid residues in the predicted mature region of Ci-META4, and tested it with regard to its antimicrobial activity. As a result, we confirmed that the synthetic peptide exhibited potent antimicrobial activity against Gram (+) and (−) bacteria, while evidencing no hemolytic activity toward human erythrocytes.     

Membrane Structure Correlates to Function of LLP2 on the Cytoplasmic Tail of HIV-1 gp41 Protein

Mutation studies previously showed that the lentivirus lytic peptide (LLP2) sequence of the cytoplasmic C-terminal tail of the HIV-1 gp41 envelope protein inhibited viral-initiated T-cell death and T-cell syncytium formation, at which time in the HIV life cycle the gp41 protein is embedded in the T-cell membrane. In striking contrast, the mutants did not affect virion infectivity, during which time the gp41 protein is embedded in the HIV envelope membrane. To examine the role of LLP2/membrane interactions, we applied synchrotron x-radiation to determine structure of hydrated membranes. We focused on WT LLP2 peptide (+3 charge) and MX2 mutant (−1 charge) with membrane mimics for the T-cell and the HIV-1 membranes. To investigate the influence of electrostatics, cholesterol content, and peptide palmitoylation, we also studied three other LLP2 variants and HIV-1 mimics without negatively charged lipids or cholesterol as well as extracted HIV-1 lipids. All LLP2 peptides bound strongly to T-cell membrane mimics, as indicated by changes in membrane structure and bending. In contrast, none of the weakly bound LLP2 variants changed the HIV-1 membrane mimic structure or properties. This correlates well with, and provides a biophysical basis for, previously published results that reported lack of a mutant effect in HIV virion infectivity in contrast to an inhibitory effect in T-cell syncytium formation. It shows that interaction of LLP2 with the T-cell membrane modulates biological function.     

Nucleotide sequence of a cDNA clone encoding the precursor of the peridinin-chlorophyll a-binding protein from the dinoflagellate Symbiodinium sp.

mRNA from the dinoflagellate Symbiodinium sp. isolated from the staghorn coral Acropora formosa was used for the construction of cDNA libraries. A cDNA clone was identified which encoded the precursor of peridinin-chlorophyll a-binding protein (PCP), including a 52 amino acid transit peptide and the 313 amino acid mature protein. The deduced amino acid sequence clearly contains an internal duplication, implying that amongst dinoflagellates the M _r 35 000 form of PCP has arisen by duplication and fusion of genes encoding the M _r 15 000 form. This is the first reported sequence of a dinoflagellate light-harvesting protein. The anatomy of the mature protein and the transit peptide are discussed.Abbreviations PCP peridinin-chlorophyll a-binding protein; cab, chlorophyll a/b-binding protein - LHC light-harvesting complex - FCP fucoxanthin-chlorophyll a/c-binding protein     

Kinetics of the unfolding-folding transition of Bacillus subtilislevansucrase precursor

The reversible folding-unfolding transition of mature and precursor forms of Bacillus subtilis levansucrase were compared under physiological conditions of pH and temperature. The time constant of the folding reaction was not modified by the presence of the signal sequence and the precursor in the native form was slightly more resistant to the denaturing action of urea. However, the folding pathway could be different for each protein since a domain of the mature levansucrase underwent an independent transition which is not observed during the renaturation process of prelevansucrase.     

GPI锚定蛋白前体C-端附着信号的疏水性决定其内质网相关蛋白质降解途径

目前,已知超过150种糖基磷脂酰肌醇锚定蛋白(glycosylphosphatidylinositol anchored protein, GPI-anchored protein)在哺乳动物细胞中表达,并参与免疫识别、细胞通讯与信号转导等多种生理过程。当蛋白质无法被GPI修饰时,前体蛋白质通过内质网相关蛋白质降解(endoplasmic reticulum associated degradation, ERAD)途径降解。然而,GPI锚定蛋白ERAD的降解机制尚不清楚。为了探究GPI锚定蛋白前体的ERAD途径的具体机制,本研究敲除人胚胎肾细胞293细胞株(HEK293)的GPI转酰胺酶复合物亚基PIGS基因,进而敲除E3泛素连接酶HRD1和GP78基因,之后随机在PIGS-KO,PIGS-HRD1-KO和PIGS-GP78-KO过表达16种GPI锚定蛋白质(以亲本PIGS-KO细胞株作为对照组),Western印迹结果证明,GPI锚定蛋白前体在细胞株PIGS-HRD1-KO中的蛋白质积累量(I_PHK)和PIGS-GP78-KO中的蛋白质积累量(I_PG...     

Nucleotide sequence and chromosomal location of Cab11 and Cab12, the genes for the fourth polypeptide of the photosystem I light-harvesting antenna (LHCI)

Tryptic peptide sequences from the 22 kDa polypeptide of tomato LHCI were used to construct a probe for gene cloning. The two genes cloned, cab11 and cab12, encode proteins of 251 and 250 residues that are 88% identical in overall amino acid sequence and 93% identical in the deduced mature protein. Each gene is present in a single copy per haploid genome; cab11 on chromosome 3 and cab12 on chromosome 6, and each has 2 introns located in similar positions to introns in other members of the Chl a/b-binding (CAB) protein gene family. Comparison of the amino acid sequences of LHCI, LHCII, CP29 and CP24 polypeptides confirms that all CABs share two regions of very high similarity which include the first and third transmembrane helices and the stroma-exposed sequences preceding them. However, near the N-terminus and between the conserved regions, the LHCI polypeptides have sequence motifs which appear to be PSI-specific.     

Characterization of the ush gene of Escherichia coli and its protein products

The Escherichia coli ush gene has been subcloned and the coding sequence delineated using BAL31 nuclease digestion. Synthesis of proteins encoded by the ush gene have been examined in "maxicells"; two proteins are made, one of which corresponds in Mr (61000) to purified uridine diphosphoglucose hydrolase and the other, less abundant, has an Mr of 43 000. A deletion at the 3' end of the gene introduced by restriction endonuclease digestion, results in the synthesis of a truncated protein of the expected Mr of about 43 000. Precursors of all these proteins are observed in maxicells under conditions known to inhibit processing of secreted proteins. Whereas the precursor of the major ush-encoded protein is retained in the cytoplasm-plus-membrane fraction, unexpectedly the precursor of the truncated protein is secreted. The mature forms of both the normal and truncated proteins are secreted.     

Characterization of the pColV-K30 encoded cloacin DF13/aerobactin outer membrane receptor protein of Escherichia coli; isolation and purification of the protein and analysis of its nucleotide sequence and primary structure

Abstract The cloacin DF13/aerobactin receptor protein from Escherichia coli (pFS8) and from Klebsiella edwardsii were isolated by repeated Triton X-100 extractions and purified by affinity chromatography. Both receptor proteins ran as a single protein band on SDS-PAGE. Their apparent M_r values were 74 000 and 76 000, respectively. The binding constants of the purified receptor proteins from E. coli (pFS8) and K. edwardsii and cloacin DF13 were determined. Values of 2.0 × 10⁸ M⁻¹ and 1.0 × 10⁹ M⁻¹, respectively, were found.
The nucleotide sequence of the pColV-K30 gene, contained on pFS8 and encoding the cloacin DF13/aerobactin receptor protein, was determined and the primary structure of the protein as well as its secondary structure were deduced. The results revealed that the pColV-K30-specified receptor protein might be synthesized as a precursor, with a signal sequence of 25 amino acid residues. The mature protein has an M_r of 77 345.     

Molecular cloning and nucleotide sequence of cDNA for sporamin,the major soluble protein of sweet potato tuberous roots

Summary Sporamin accounts for more than 80% of the total soluble proteins of tuberous roots of sweet potato, but very little, if any, in other tissues of the same plant. In vitro translation of RNA fractions from the tuberous roots in wheat germ extract and subsequent immunoprecipitation with the antibody to sporamin indicated that this protein is synthesized by membrane-bound polysomes as a precursor 4 000 daltons larger than the mature protein. A cDNA expression library was constructed from the total poly(A)⁺ RNA from the tuberous roots by a vector-primer method, and an essentially full-length cDNA clone for the sporamin mRNA was selected by direct immunological screening of the colonies. Northern blot analysis showed that sporamin mRNA is approximately 950 nucleotides in length and is specifically present in tuberous roots and very little, if any, in leaves, petioles and non-tuberous roots. Nucleotide sequence of the cDNA predicts a 37 amino acid extension in the precursor at the amino-terminus of the mature protein.     

Identification of N-terminal methionine in the precursor of immunoglobulin light chain. Initiation of translation of messenger ribonucleic acid in plants and animals.

The proteins programmed in the wheat-germ cell-free system by the mRNA coding for the MOPC-321 mouse myeloma L (light) chain were labelled with [35S]methionine, [4,5-3H]leucine or [3-3H]serine, and were subjected to amino acid-sequence analyses. Over 95% of the total cell-free product was sequenced as one homogeneous protein, which corresponds to the precursor of the L-chain protein. In the precursor, 20 amino acid residues precede the N-terminus of the mature protein. This extra piece contains one methionine residue at the N-terminus, one serine residue at position 18, and six leucine residues, which are clustered in two triplets at positions 6, 7, 8 and 11, 12, 13. The identification of methionine at the N-terminus of the precursor is in agreement with the evidence showing that unblocked methionine is the initiator residue for protein synthesis in eukaryotes. The absence of methionine at position 20, which precedes the N-terminal residue of the mature protein, suggests that myeloma cells synthesize the precursor. However, within the cell the precursor should be rapidly processed to the mature L chain, since precursor molecules have not yet been found in the intact animal. The abundance (30%) of leucine residues indicates that the extra-piece moiety is quite hydrophobic. The extra piece of the MOPC-321 L-chain precursor synthesized with the aid of the Krebs II ascites cell-free system is of identical size and it has the same leucine sequence [Schechter et al. (1975) Science 188, 160-162]. This indicates that cell-free systems derived from the plant and animal kingdom initiate mRNA translation from the same point. It is shown that the amino acid sequence of minute amounts of a highly labelled protein (0.1 pmol) can be faithfully determined in the presence of a large excess (over 2000 000-fold) of unrelated non-radioactive proteins.     

Reconstitution of the spinach oxygen-evolving complex with recombinant Arabidopsis manganese-stabilizing protein

The psbO gene of cyanobacteria, green algae and higher plants encodes the precursor of the 33 kDa manganese-stabilizing protein (MSP), a water-soluble subunit of photosystem II (PSII). Using a pET-T7 cloning/expression system, we have expressed in Escherichia coli a full-length cDNA clone of psbO from Arabidopsis thaliana. Upon induction, high levels of the precursor protein accumulated in cells grown with vigorous aeration. In cells grown under weak aeration, the mature protein accumulated upon induction. In cells grown with moderate aeration, the ratio of precursor to mature MSP decreased as the optical density at induction increased. Both forms of the protein accumulated as inclusion bodies from which the mature protein could be released under mildly denaturing conditions that did not release the precursor. Renatured Arabidopsis MSP was 87% as effective as isolated spinach MSP in restoring O₂ evolution activity to MSP-depleted PSII membranes from spinach; however, the heterologous protein binds to spinach PSIIs with about half the affinity of the native protein. We also report a correction to the previously published DNA sequence of Arabidopsis psbO (Ko et al., Plant Mol Biol 14 (1990) 217–227).     

Primary structure of a chitinase-encoding gene (chi1) from the filamentous fungus Aphanocladium album: similarity to bacterial chitinases.

Chitinase 1 (Chil) is the major extracellular chitinase from the hyperparasitic fungus, Aphanocladium album. We determined the complete sequence of the chromosomal and cDNA copies of the structural gene (chi1) coding for Chil. The coding region is interrupted by three short introns (55, 53 and 49 bp long). Chil is 423 aa long and begins with a stretch of 34 aa not found in the mature protein. The Chil sequence presents overall similarities with bacterial chitinases from Serratia marcescens and Bacillus circulans. Compared with other chitinases, A. album Chi1 has only two short similarity regions (12 and 8 aa long), which are also found in bacterial, yeast and some plant chitinases.     

A novel chlorophyll a/b binding (Cab) protein gene from petunia which encodes the lower molecular weight Cab precursor protein.

The 16 petunia Cab genes which have been characterized are all closely related at the nucleotide sequence level and they encode Cab precursor polypeptides which are similar in sequence and length. Here we describe a novel petunia Cab gene which encodes a unique Cab precursor protein. This protein is a member of the smallest class of Cab precursor proteins for which no gene has previously been assigned in petunia or any other species. The features of this Cab precursor protein are that it is shorter by 2-3 amino acids than the formerly characterized Cab precursors, its transit peptide sequence is unrelated, and the mature polypeptide is significantly diverged at the functionally important N terminus from other petunia Cab proteins. Gene structure also discriminates this gene which is the only intron containing Cab gene in petunia genomic DNA.     

Role of the signal sequence in proteorhodopsin biogenesis in E. coli

Blue-absorbing proteorhodopsin (BPR) from marine bacteria is a retinal-bound, light-activated, outwards proton transporter containing seven α-helical transmembrane segments (TMS). It is synthesized as a precursor species (pre-BPR) with a predicted N-terminal signal sequence that is cleaved to yield the mature protein. While optimizing the production of BPR in Escherichia coli to facilitate the construction of bioprotonic devices, we observed significant pre-BPR accumulation in the inner membrane and explored signal sequence requirements and export pathway. We report here that BPR does not rely on the Sec pathway for inner membrane integration, and that although it greatly enhances yields, its signal sequence is not necessary to obtain a functional product. We further show that an unprocessable version of pre-BPR obtained by mutagenesis of the signal peptidase I site exhibits all functional attributes of the wild-type protein and has the advantage of being produced at higher levels. Our results are consistent with the BPR signal sequence being recognized by the signal recognition particle (SRP; a protein that orchestrates the cotranslational biogenesis of inner membrane proteins) and serving as a beneficial “pro” domain rather than a traditional secretory peptide.