scholarly journals Chloroplast transit peptides often require downstream unstructured sequence in Chlamydomonas reinhardtii

2021 ◽  
Author(s):  
Oliver D Caspari
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Cleavage Site ◽  
Mature Protein ◽  
Terminal Sequence ◽  
Subcellular Targeting ◽  
Chloroplast Targeting ◽  
Chloroplast Proteins ◽  
Transit Peptides ◽  
Additional Sequence ◽  
Sequence Stretch

The N-terminal sequence stretch that defines subcellular targeting for most nuclear encoded chloroplast proteins is usually considered identical to the sequence that is cleaved upon import. Yet here this study shows that for nine out of ten tested Chlamydomonas chloroplast transit peptides, additional sequence past the cleavage site is required to enable chloroplast targeting. Using replacements of native post-cleavage residues with alternative sequences points to a role for unstructured sequence at mature protein N-termini.

scholarly journals atToc159 is a selective transit peptide receptor for the import of nucleus-encoded chloroplast proteins

2004 ◽  
Vol 165 (3) ◽  
pp. 323-334 ◽  
Author(s):  
Matthew D. Smith ◽  
Caleb M. Rounds ◽  
Fei Wang ◽  
Kunhua Chen ◽  
Meshack Afitlhile ◽  
...  
Keyword(s):  
Plant Cell ◽  
Chloroplast Development ◽  
Transit Peptide ◽  
Chloroplast Biogenesis ◽  
Cross Link ◽  
Peptide Receptor ◽  
Chloroplast Proteins ◽  
Transit Peptides ◽  
Import Receptor

The members of the Toc159 family of GTPases act as the primary receptors for the import of nucleus-encoded preproteins into plastids. Toc159, the most abundant member of this family in chloroplasts, is required for chloroplast biogenesis (Bauer, J., K. Chen, A. Hiltbunner, E. Wehrli, M. Eugster, D. Schnell, and F. Kessler. 2000. Nature. 403:203–207) and has been shown to covalently cross-link to bound preproteins at the chloroplast surface (Ma, Y., A. Kouranov, S. LaSala, and D.J. Schnell. 1996. J. Cell Biol. 134:1–13; Perry, S.E., and K. Keegstra. 1994. Plant Cell. 6:93–105). These reports led to the hypothesis that Toc159 functions as a selective import receptor for preproteins that are required for chloroplast development. In this report, we provide evidence that Toc159 is required for the import of several highly expressed photosynthetic preproteins in vivo. Furthermore, we demonstrate that the cytoplasmic and recombinant forms of soluble Toc159 bind directly and selectively to the transit peptides of these representative photosynthetic preproteins, but not representative constitutively expressed plastid preproteins. These data support the function of Toc159 as a selective import receptor for the targeting of a set of preproteins required for chloroplast biogenesis.

Identification and characterisation of a novel inorganic carbon acquisition gene, CIA7, from an insertional mutant of Chlamydomonas reinhardtii

2008 ◽  
Vol 35 (5) ◽  
pp. 373 ◽  
Author(s):  
Ruby A. Ynalvez ◽  
James V. Moroney
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Metal Ion ◽  
Inorganic Carbon ◽  
Antibiotic Resistance Gene ◽  
Inverse Pcr ◽  
Chloroplast Targeting ◽  
Calculated Molecular Mass ◽  
Prediction Program ◽  
Metal Transporter ◽  
Low Co2

Chlamydomonas reinhardtii is a unicellular eukaryotic alga which possesses a CO2-concentrating mechanism (CCM) that enables it to grow at low CO2 concentrations. Previously, insertional mutants were generated to enable isolation of inorganic carbon transporters and other proteins that might be essential for a functional CCM. These mutants have an antibiotic resistance gene that encodes a protein that binds to Zeocin inhibiting Zeocin’s DNA strand cleavage activity. The DNA flanking the BleR insert of one of the high CO2 requiring strains, named cia7, was cloned with inverse-PCR and sequenced. Sequence analysis showed homology to conserved bacterial proteins of unknown function, but there were no ESTs in this region of the genome. However, the presence of a gene was established by PCR and RLM-RACE. CIA7 was found to have four exons and the BleR insert was in the fourth exon. CIA7 encodes a protein of 104 amino acids with a calculated molecular mass of 11.3 kDa. Based on the ChloroP prediction program, the protein is predicted to have a chloroplast targeting signal. Complementation analyses results showed possible partially rescued mutants, and RNAi showed several transformants with a sick on low CO2 phenotype with reduced expression of CIA7. These results suggest that CIA7 is a gene that facilitates growth in C. reinhardtii under low CO2 conditions. One possible role of CIA7 would be in the delivery or storage of a metal ion. It may play a potential role as either a domain of a metal transporter or as a metallochaperone.

scholarly journals Arabidopsis Nuclear-Encoded Plastid Transit Peptides Contain Multiple Sequence Subgroups with Distinctive Chloroplast-Targeting Sequence Motifs

2008 ◽  
Vol 20 (6) ◽  
pp. 1603-1622 ◽  
Author(s):  
Dong Wook Lee ◽  
Jong Kyoung Kim ◽  
Sumin Lee ◽  
Seungjin Choi ◽  
Sanguk Kim ◽  
...  
Keyword(s):  
Sequence Motifs ◽  
Multiple Sequence ◽  
Chloroplast Targeting ◽  
Transit Peptides

scholarly journals Stromal Processing Peptidase Binds Transit Peptides and Initiates Their Atp-Dependent Turnover in Chloroplasts

1999 ◽  
Vol 147 (1) ◽  
pp. 33-44 ◽  
Author(s):  
Stefan Richter ◽  
Gayle K. Lamppa
Keyword(s):  
Binding Assay ◽  
Specific Binding ◽  
Transit Peptide ◽  
Binding Motif ◽  
Mature Protein ◽  
Sequence Of Events ◽  
Mitochondrial Processing Peptidase ◽  
Transit Peptides ◽  
Processing Peptidase ◽  
Zinc Binding Motif

A stromal processing peptidase (SPP) cleaves a broad range of precursors targeted to the chloroplast, yielding proteins for numerous biosynthetic pathways in different compartments. SPP contains a signature zinc-binding motif, His-X-X-Glu-His, that places it in a metallopeptidase family which includes the mitochondrial processing peptidase. Here, we have investigated the mechanism of cleavage by SPP, a late, yet key event in the import pathway. Recombinant SPP removed the transit peptide from a variety of precursors in a single endoproteolytic step. Whereas the mature protein was immediately released, the transit peptide remained bound to SPP. SPP converted the transit peptide to a subfragment form that it no longer recognized. We conclude that SPP contains a specific binding site for the transit peptide and additional proteolysis by SPP triggers its release. A stable interaction between SPP and an intact transit peptide was directly demonstrated using a newly developed binding assay. Unlike recombinant SPP, a chloroplast extract rapidly degraded both the transit peptide and subfragment. A new degradative activity, distinguishable from SPP, was identified that is ATP- and metal-dependent. Our results indicate a regulated sequence of events as SPP functions during precursor import, and demonstrate a previously unrecognized ATP-requirement for transit peptide turnover.

scholarly journals A Cytochrome b 5 -Containing Plastid-Located Fatty Acid Desaturase from Chlamydomonas reinhardtii

2012 ◽  
Vol 11 (7) ◽  
pp. 856-863 ◽  
Author(s):  
Simone Zäuner ◽  
Wibke Jochum ◽  
Tara Bigorowski ◽  
Christoph Benning
Keyword(s):  
Fatty Acid ◽  
Chlamydomonas Reinhardtii ◽  
Fluorescent Protein ◽  
Acyl Chain ◽  
Transit Peptide ◽  
Primary Electron ◽  
Chloroplast Targeting ◽  
Content Type ◽  
Cell Extracts

ABSTRACT Monogalactosyldiacylglycerol (MGDG) in Chlamydomonas reinhardtii and other green algae contains hexadeca-4,7,10,13-tetraenoic acid (16:4) in the glycerol sn- 2 position. While many genes necessary for the introduction of acyl chain double bonds have been functionally characterized, the Δ4-desaturase remained unknown. Using a phylogenetic comparison, a candidate gene encoding the MGDG-specific Δ4-desaturase from Chlamydomonas (CrΔ4FAD) was identified. CrΔ4FAD shows all characteristic features of a membrane-bound desaturase, including three histidine boxes and a transit peptide for chloroplast targeting. But it also has an N-terminal cytochrome b 5 domain, distinguishing it from other known plastid desaturases. Cytochrome b 5 is the primary electron donor for endoplasmic reticulum (ER) desaturases and is often fused to the desaturase domain in desaturases modifying the carboxyl end of the acyl group. Difference absorbance spectra of the recombinant cytochrome b 5 domain of CrΔ4FAD showed that it is functional in vitro . Green fluorescent protein fusions of CrΔ4FAD localized to the plastid envelope in Chlamydomonas . Interestingly, overproduction of CrΔ4FAD in Chlamydomonas not only increased levels of 16:4 acyl groups in cell extracts but specifically increased the total amount of MGDG. Vice versa, the amount of MGDG was lowered in lines with reduced levels of CrΔ4FAD. These data suggest a link between MGDG molecular species composition and galactolipid abundance in the alga, as well as a specific function for this fatty acid in MGDG.

scholarly journals Purification and biosynthesis of a derepressible periplasmic arylsulfatase from Chlamydomonas reinhardtii.

1988 ◽  
Vol 106 (1) ◽  
pp. 29-37 ◽  
Author(s):  
E L de Hostos ◽  
R K Togasaki ◽  
A Grossman
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Molecular Mass ◽  
Culture Medium ◽  
Periplasmic Protein ◽  
Mature Protein ◽  
Ph Optimum ◽  
Unicellular Green Alga ◽  
Arylsulfatase Activity ◽  
A Cell ◽  
Monospecific Antibodies

The unicellular green alga Chlamydomonas reinhardtii responds to sulfate deprivation by producing an arylsulfatase (Lien, T., and O. Schreiner. 1975. Biochim. Biophys. Acta. 384:168-179; Schreiner, O., 1975. Biochim. Biophys. Acta. 384:180-193) and by developing the capacity to transport sulfate more rapidly (our unpublished data). The arylsulfatase activity, detectable 3 h after the transfer of the cells to low sulfate medium (less than or equal to 10 microM sulfate), is a periplasmic protein released into the culture medium by cw15, a cell wall-less mutant of C. reinhardtii. We have purified the derepressible arylsulfatase to homogeneity and have raised monospecific antibodies to it. The protein monomer (67.6 kD) associates into a dimer, and the enzyme activity shows an alkaline pH optimum and a Km of 0.3 mM for p-nitrophenylsulfate. Studies focused on arylsulfatase biosynthesis demonstrate that it is glycosylated and synthesized as a higher molecular mass precursor. The mature protein contains complex N-linked oligosaccharides and the primary translation product has an apparent molecular mass approximately 5 kD larger than the deglycosylated monomer. Since translatable RNA encoding the arylsulfatase can only be detected in cells after sulfate starvation, it is likely that accumulation of the enzyme is regulated at the level of transcription, although posttranscriptional processes may also be involved.

scholarly journals A conserved cleavage-site motif in chloroplast transit peptides

FEBS Letters ◽  
1990 ◽  
Vol 261 (2) ◽  
pp. 455-458 ◽  
Author(s):  
Ylva Gavel ◽  
Gunnar von Heijne
Keyword(s):  
Cleavage Site ◽  
Transit Peptides

scholarly journals Membrane lipid biosynthesis in Chlamydomonas reinhardtii: expression and characterization of CTP:phosphoethanolamine cytidylyltransferase

2004 ◽  
Vol 382 (1) ◽  
pp. 51-57 ◽  
Author(s):  
Wenyu YANG ◽  
Catherine B. MASON ◽  
Steve V. POLLOCK ◽  
Tracey LAVEZZI ◽  
James V. MORONEY ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chlamydomonas Reinhardtii ◽  
Translational Regulation ◽  
Mrna Level ◽  
Total Activity ◽  
Membrane Lipid ◽  
Peptide Sequence ◽  
Marker Enzyme ◽  
Amino Acid Residues ◽  
Subcellular Targeting

CTP:phosphoethanolamine cytidylyltransferase (ECT) is considered to be the regulatory enzyme in the CDP-ethanolamine pathway of phosphatidylethanolamine (PE) biosynthesis. The ECT cDNA of Chlamydomonas reinhardtii encodes a protein of 443 amino acid residues, which is longer than the same protein in yeast, rat or human. The translated product of cloned cDNA was expressed as a fusion protein in Escherichia coli, and was shown to have ECT activity. The deduced amino acid sequence has 41% identity with that of human or rat, and 30% with yeast. The ECT protein has a repetitive internal sequence in its N- and C-terminal halves and a signature peptide sequence, RTXGVSTT, typical of the cytidylyltransferase family. The first 70 amino acid residues do not match the N-terminal part of the cytidylyltransferases from other organisms, and we hypothesize that it is a subcellular targeting signal to mitochondria. ECT and organelle marker enzyme assays showed that the total activity of ECT correlates well with that of fumarase, a marker enzyme for mitochondria. Northern blots showed an increase in mRNA abundance during reflagellation, indicating a possibility of transcriptional regulation. A notable change in the enzyme activity in C. reinhardtii cells was observed during the cell cycle, increasing during the dark and then decreasing during the light period, while the mRNA level did not alter, providing evidence for post-translational regulation.

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