scholarly journals Chloroplast chaperonin-mediated targeting of a thylakoid membrane protein

2020 ◽  
Author(s):  
Laura Klasek ◽  
Kentaro Inoue ◽  
Steven M. Theg
Keyword(s):  
Membrane Protein ◽  
Thylakoid Membrane ◽  
Protein Targeting ◽  
Transmembrane Proteins ◽  
Signal Peptidase ◽  
Aqueous Environment ◽  
Type I ◽  
Isolated Chloroplasts

AbstractPost-translational protein targeting requires chaperone assistance to direct insertion-competent proteins to integration pathways. Chloroplasts integrate nearly all thylakoid transmembrane proteins post-translationally, but mechanisms in the stroma that assist their insertion remain largely undefined. Here, we investigated how the chloroplast chaperonin (Cpn60) facilitated the thylakoid integration of Plastidic type I signal peptidase 1 (Plsp1) using in vitro targeting assays. Cpn60 bound Plsp1 in the stroma. In isolated chloroplasts, the membrane integration of imported Plsp1 correlated with its dissociation from Cpn60. When the Plsp1 residues that interacted with Cpn60 were removed, Plsp1 did not integrate into the membrane. These results suggested Cpn60 was an intermediate in Plsp1’s thylakoid targeting. In isolated thylakoids, the integration of Plsp1 decreased if Cpn60 was present in excess of cpSecA1, the stromal motor of the cpSec1 translocon which inserts unfolded Plsp1 into the thylakoid. An excess of cpSecA1 favored integration. Introducing Cpn60’s obligate substrate RbcL displaced Cpn60-bound Plsp1; then, the released Plsp1 exhibited increased accessibility to cpSec1. These in vitro targeting experiments support a model in which Cpn60 captures and then releases insertion-competent Plsp1, while cpSecA1 recognizes free Plsp1 for integration. Thylakoid transmembrane proteins transiting the stroma can interact with Cpn60 to shield from the aqueous environment.One-sentence summaryThe chloroplast chaperonin captures and releases Plastidic type I signal peptidase 1 during its targeting to the thylakoid membrane.

scholarly journals P13, an Integral Membrane Protein of Borrelia burgdorferi, Is C-Terminally Processed and Contains Surface-Exposed Domains

2001 ◽  
Vol 69 (5) ◽  
pp. 3323-3334 ◽  
Author(s):  
Laila Noppa ◽  
Yngve Östberg ◽  
Marija Lavrinovicha ◽  
Sven Bergström
Keyword(s):  
Membrane Protein ◽  
Borrelia Burgdorferi ◽  
Gene Family ◽  
Integral Membrane Protein ◽  
Sensu Stricto ◽  
In Vitro Translation ◽  
Signal Peptidase ◽  
In Vitro Cultivation ◽  
Type I

ABSTRACT To elucidate antigens present on the bacterial surface ofBorrelia burgdorferi sensu lato that may be involved in pathogenesis, we characterized a protein, P13, with an apparent molecular mass of 13 kDa. The protein was immunogenic and was expressed in large amounts during in vitro cultivation compared to other known antigens. An immunofluorescence assay, immunoelectron microscopy, and protease sensitivity assays indicated that P13 is surface exposed. The deduced sequence of the P13 peptide revealed a possible signal peptidase type I cleavage site, and computer analysis predicted that P13 is an integral membrane protein with three transmembrane-spanning domains. Mass spectrometry, in vitro translation, and N- and C-terminal amino acid sequencing analyses indicated that P13 was posttranslationally processed at both ends and modified by an unknown mechanism. Furthermore, p13 belongs to a gene family with five additional members in B. burgdorferi sensu stricto. The p13 gene is located on the linear chromosome of the bacterium, in contrast to five paralogous genes, which are located on extrachromosomal plasmids. The size of the p13 transcript was consistent with a monocistronic transcript. This new gene family may be involved in functions that are specific for this spirochete and its pathogenesis.

scholarly journals Lipid Chaperoning of a Thylakoid Protease Whose Stability is Modified by the Protonmotive Force

2019 ◽  
Author(s):  
Lucas J. McKinnon ◽  
Jeremy Fukushima ◽  
Kentaro Inoue ◽  
Steven M. Theg
Keyword(s):  
Disulfide Bond ◽  
Thylakoid Membrane ◽  
Bond Formation ◽  
Disulfide Bridge ◽  
Signal Peptidase ◽  
Common Mechanism ◽  
Protonmotive Force ◽  
Type I ◽  
Disulfide Bond Formation

AbstractProtein folding is a complex cellular process often assisted by chaperones but can also be facilitated by interactions with lipids. Disulfide bond formation is a common mechanism to stabilize a protein. This can help maintain functionality amidst changes in the biochemical milieu which are especially common across energy-transducing membranes. Plastidic Type I Signal Peptidase 1 (Plsp1) is an integral thylakoid membrane signal peptidase which requires an intramolecular disulfide bond for in vitro activity. We have investigated the interplay between disulfide bond formation, lipids, and pH in the folding and activity of Plsp1. By combining biochemical approaches with a genetic complementation assay, we provide evidence that interactions with lipids in the thylakoid membrane have chaperoning activity towards Plsp1. Further, the disulfide bridge appears to prevent an inhibitory conformational change resulting from proton motive force-mimicking pH conditions. Broader implications related to the folding of proteins in energy-transducing membranes are discussed.

scholarly journals In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules.

1987 ◽  
Vol 105 (2) ◽  
pp. 659-668 ◽  
Author(s):  
T L Burgess ◽  
C S Craik ◽  
L Matsuuchi ◽  
R B Kelly
Keyword(s):  
Signal Peptide ◽  
Secretory Pathway ◽  
Protein Targeting ◽  
Secretory Granules ◽  
Tumor Cell Line ◽  
Signal Peptidase ◽  
Secretory Proteins ◽  
In Vitro Mutagenesis ◽  
Regulated Secretory Pathway

The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.

scholarly journals The SH3 domain of the Saccharomyces cerevisiae peroxisomal membrane protein Pex13p functions as a docking site for Pex5p, a mobile receptor for the import PTS1-containing proteins.

1996 ◽  
Vol 135 (1) ◽  
pp. 97-109 ◽  
Author(s):  
Y Elgersma ◽  
L Kwast ◽  
A Klein ◽  
T Voorn-Brouwer ◽  
M van den Berg ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Protein ◽  
Protein Import ◽  
Sh3 Domain ◽  
Type I ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein ◽  
Peroxisomal Targeting ◽  
Src Homology

We identified a Saccharomyces cerevisiae peroxisomal membrane protein, Pex13p, that is essential for protein import. A point mutation in the COOH-terminal Src homology 3 (SH3) domain of Pex13p inactivated the protein but did not affect its membrane targeting. A two-hybrid screen with the SH3 domain of Pex13p identified Pex5p, a receptor for proteins with a type I peroxisomal targeting signal (PTS1), as its ligand. Pex13p SH3 interacted specifically with Pex5p in vitro. We determined, furthermore, that Pex5p was mainly present in the cytosol and only a small fraction was associated with peroxisomes. We therefore propose that Pex13p is a component of the peroxisomal protein import machinery onto which the mobile Pex5p receptor docks for the delivery of the selected PTS1 protein.

scholarly journals A Putative Bacterial ABC Transporter Circumvents the Essentiality of Signal Peptidase

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
J. Hiroshi Morisaki ◽  
Peter A. Smith ◽  
Shailesh V. Date ◽  
Kimberly K. Kajihara ◽  
Chau Linda Truong ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Abc Transporter ◽  
Signal Peptide ◽  
Transcriptional Repressor ◽  
Signal Peptidase ◽  
Clinical Strain ◽  
Type I ◽  
Atpase Gene ◽  
Associated Proteins

ABSTRACT The type I signal peptidase of Staphylococcus aureus , SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro / cI , a homolog of the lambda phage transcriptional repressor cro . These cro / cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB . Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo . IMPORTANCE The type I signal peptidase of Staphylococcus aureus (SpsB) enables the secretion of numerous proteins by cleavage of the signal peptide. We synthesized an SpsB inhibitor with potent activity against various clinical S. aureus strains. The predominant S. aureus strain USA300 develops resistance to this inhibitor by mutations in a novel transcriptional repressor ( cro / cI ), causing overexpression of a putative ABC transporter. This mechanism promotes the cleavage and secretion of various proteins independently of SpsB and compensates for the requirement of SpsB for viability in vitro . However, bacteria overexpressing the ABC transporter and lacking SpsB secrete reduced levels of virulence-associated proteins and are unable to infect mice. This study describes a bacterial resistance mechanism that provides novel insights into the biology of bacterial secretion.

scholarly journals Broadening the Spectrum of β-Lactam Antibiotics through Inhibition of Signal Peptidase Type I

2012 ◽  
Vol 56 (9) ◽  
pp. 4662-4670 ◽  
Author(s):  
Alex G. Therien ◽  
Joann L. Huber ◽  
Kenneth E. Wilson ◽  
Patrick Beaulieu ◽  
Alexandre Caron ◽  
...  
Keyword(s):  
Treatment Options ◽  
Signal Peptidase ◽  
Type I ◽  
New Agents ◽  
Content Type ◽  
Cyclic Depsipeptide ◽  
Serious Infections ◽  
Synthetic Derivative

ABSTRACTThe resistance of methicillin-resistantStaphylococcus aureus(MRSA) to all β-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of β-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem bothin vitroandin vivowith potent efficacy. Thein vitroactivity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to β-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with β-lactams by preventing the signal peptidase-mediated secretion of proteins required for β-lactam resistance. Combinations of SpsB inhibitors and β-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to β-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.

Evaluation of the type I signal peptidase as antibacterial target for biofilm-associated infections of Staphylococcus epidermidis

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3719-3729 ◽  
Author(s):  
Katrijn Bockstael ◽  
Nick Geukens ◽  
Lieve Van Mellaert ◽  
Piet Herdewijn ◽  
Jozef Anné ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Functional Activity ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Antimicrobial Treatment ◽  
Signal Peptidase ◽  
Temperature Sensitive ◽  
Type I

The development of antibacterial resistance is inevitable and is a major concern in hospitals and communities. Moreover, biofilm-grown bacteria are less sensitive to antimicrobial treatment. In this respect, the Gram-positive Staphylococcus epidermidis is an important source of nosocomial biofilm-associated infections. In the search for new antibacterial therapies, the type I signal peptidase (SPase I) serves as a potential target for development of antibacterials with a novel mode of action. This enzyme cleaves off the signal peptide from secreted proteins, making it essential for protein secretion, and hence for bacterial cell viability. S. epidermidis encodes three putative SPases I (denoted Sip1, Sip2 and Sip3), of which Sip1 lacks the catalytic lysine. In this report, we investigated the active S. epidermidis SPases I in more detail. Sip2 and Sip3 were found to complement a temperature-sensitive Escherichia coli lepB mutant, demonstrating their in vivo functional activity. In vitro functional activity of purified Sip2 and Sip3 proteins and inhibition of their activity by the SPase I inhibitor arylomycin A2 were further illustrated using a fluorescence resonance energy transfer (FRET)-based assay. Furthermore, we demonstrated that SPase I not only is an attractive target for development of novel antibacterials against free-living bacteria, but also is a feasible target for biofilm-associated infections.

scholarly journals Molecular Analysis of Phr Peptide Processing in Bacillus subtilis

2003 ◽  
Vol 185 (16) ◽  
pp. 4861-4871 ◽  
Author(s):  
Sophie Stephenson ◽  
Christian Mueller ◽  
Min Jiang ◽  
Marta Perego
Keyword(s):  
Bacillus Subtilis ◽  
Response Regulator ◽  
Signal Peptidase ◽  
Type I ◽  
Peptidase Activity ◽  
Amino Terminal ◽  
Processing Event ◽  
Signal Peptidases

ABSTRACT In Bacillus subtilis, an export-import pathway regulates production of the Phr pentapeptide inhibitors of Rap proteins. Processing of the Phr precursor proteins into the active pentapeptide form is a key event in the initiation of sporulation and competence development. The PhrA (ARNQT) and PhrE (SRNVT) peptides inhibit the RapA and RapE phosphatases, respectively, whose activity is directed toward the Spo0F∼P intermediate response regulator of the sporulation phosphorelay. The PhrC (ERGMT) peptide inhibits the RapC protein acting on the ComA response regulator for competence with regard to DNA transformation. The structural organization of PhrA, PhrE, and PhrC suggested a role for type I signal peptidases in the processing of the Phr preinhibitor, encoded by the phr genes, into the proinhibitor form. The proinhibitor was then postulated to be cleaved to the active pentapeptide inhibitor by an additional enzyme. In this report, we provide evidence that Phr preinhibitor proteins are subject to only one processing event at the peptide bond on the amino-terminal end of the pentapeptide. This processing event is most likely independent of type I signal peptidase activity. In vivo and in vitro analyses indicate that none of the five signal peptidases of B. subtilis (SipS, SipT, SipU, SipV, and SipW) are indispensable for Phr processing. However, we show that SipV and SipT have a previously undescribed role in sporulation, competence, and cell growth.

scholarly journals A stromal protein factor maintains the solubility and insertion competence of an imported thylakoid membrane protein.

1991 ◽  
Vol 112 (4) ◽  
pp. 603-613 ◽  
Author(s):  
L A Payan ◽  
K Cline
Keyword(s):  
Membrane Protein ◽  
Thylakoid Membrane ◽  
Gel Filtration ◽  
Chloroplast Envelope ◽  
Membrane Insertion ◽  
Protein Factor ◽  
Stromal Component ◽  
Thylakoid Protein ◽  
Chloroplast Stroma

The light-harvesting chlorophyll a/b protein (LHCP) is an approximately 25,000-D thylakoid membrane protein. LHCP is synthesized in the cytosol as a precursor and must translocate across the chloroplast envelope before becoming integrally associated with the thylakoid bilayer. Previous studies demonstrated that imported LHCP traverses the chloroplast stroma as a soluble intermediate before thylakoid insertion. Here, examination of this intermediate revealed that it is a stable, discrete approximately 120,000-D species and thus either an LHCP oligomer or a complex with another component. In vitro-synthesized LHCP can be converted to a similar form by incubation with a stromal extract. The stromal component responsible for this conversion is proteinaceous as evidenced by its inactivation by heat, protease, and NEM. Furthermore, the conversion activity coelutes from a gel filtration column with a stromal protein factor(s) previously shown to be necessary for LHCP integration into isolated thylakoids. Conversion of LHCP to the 120-kD form prevents aggregation and maintains its competence for thylakoid insertion. However, conversion to this form is apparently not sufficient for membrane insertion because the isolated 120-kD LHCP still requires stroma to complete the integration process. This suggests a need for at least one more stroma-mediated reaction. Our results explain how a hydrophobic thylakoid protein remains soluble as it traverses the aqueous stroma. Moreover, they describe in part the function of the stromal requirement for insertion into the thylakoid membrane.

scholarly journals Complete maturation of the plastid protein translocation channel requires a type I signal peptidase

2005 ◽  
Vol 171 (3) ◽  
pp. 425-430 ◽  
Author(s):  
Kentaro Inoue ◽  
Amy J. Baldwin ◽  
Rebecca L. Shipman ◽  
Kyoko Matsui ◽  
Steven M. Theg ◽  
...  
Keyword(s):  
Protein Translocation ◽  
Biological Significance ◽  
Transit Peptide ◽  
Signal Peptidase ◽  
Type I ◽  
Gene Encoding ◽  
Severe Reduction ◽  
Plastid Protein ◽  
Outer Envelope Membrane

The protein translocation channel at the plastid outer envelope membrane, Toc75, is essential for the viability of plants from the embryonic stage. It is encoded in the nucleus and is synthesized with a bipartite transit peptide that is cleaved during maturation. Despite its important function, the molecular mechanism and the biological significance of the full maturation of Toc75 remain unclear. In this study, we show that a type I signal peptidase (SPase I) is responsible for this process. First, we demonstrate that a bacterial SPase I converted Toc75 precursor to its mature form in vitro. Next, we show that disruption of a gene encoding plastidic SPase I (Plsp1) resulted in the accumulation of immature forms of Toc75, severe reduction of plastid internal membrane development, and a seedling lethal phenotype. These phenotypes were rescued by the overexpression of Plsp1 complementary DNA. Plsp1 appeared to be targeted both to the envelope and to the thylakoidal membranes; thus, it may have multiple functions.

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