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S Merchant

Publications and source records attributed to S Merchant.

At least 37 records · Page 2Linked to original sources
PubMed1998

Molecular mechanisms of cytochrome c biogenesis: three distinct systems.

The past 10 years have heralded remarkable progress in the understanding of the biogenesis of c-type cytochromes. The hallmark of c-type cytochrome synthesis is the covalent ligation of haem vinyl groups to two cysteinyl residues of the apocytochrome (at a Cys-Xxx-Yyy-Cys-His signature motif). From genetic, genomic and biochemical studies, it is clear that three distinct systems have evolved in nature to assemble this ancient protein. In this review, common principles of assembly for all systems and the molecular mechanisms predicted for each system are summarized. Prokaryotes, plant mitochondria and chloroplasts use either system I or II, which are each predicted to use dedicated mechanisms for haem delivery, apocytochrome ushering and thioreduction. Accessory proteins of systems I and II co-ordinate the positioning of these two substrates at the membrane surface for covalent ligation. The third system has evolved specifically in mitochondria of fungi, invertebrates and vertebrates. For system III, a pivotal role is played by an enzyme called cytochrome c haem lyase (CCHL) in the mitochondrial intermembrane space.

Apoproteins↗
PubMed1998

Genetic analysis of chloroplast c-type cytochrome assembly in Chlamydomonas reinhardtii: One chloroplast locus and at least four nuclear loci are required for heme attachment.

Chloroplasts contain up to two c-type cytochromes, membrane-anchored cytochrome f and soluble cytochrome c6. To elucidate the post-translational events required for their assembly, acetate-requiring mutants of Chlamydomonas reinhardtii that have combined deficiencies in both plastid-encoded cytochrome f and nucleus-encoded cytochrome c6 have been identified and analyzed. For strains ct34 and ct59, where the phenotype displays uniparental inheritance, the mutations were localized to the chloroplast ccsA gene, which was shown previously to be required for heme attachment to chloroplast apocytochromes. The mutations in another eight strains were localized to the nuclear genome. Complementation tests of these strains plus three previously identified strains of the same phenotype (ac206, F18, and F2D8) indicate that the 11 ccs strains define four nuclear loci, CCS1-CCS4. We conclude that the products of the CCS1-CCS4 loci are not required for translocation or processing of the preproteins but, like CcsA, they are required for the heme attachment step during assembly of both holocytochrome f and holocytochrome c6. The ccsA gene is transcribed in each of the nuclear mutants, but its protein product is absent in ccs1 mutants, and it appears to be degradation susceptible in ccs3 and ccs4 strains. We suggest that Ccsl may be associated with CcsA in a multisubunit "holocytochrome c assembly complex," and we hypothesize that the products of the other CCS loci may correspond to other subunits.

Animals↗
PubMed1997

Molecular genetic identification of a pathway for heme binding to cytochrome b6.

Heme binding to cytochrome b6 is resistant, in part, to denaturing conditions that typically destroy the noncovalent interactions between the b hemes and their apoproteins, suggesting that one of two b hemes of holocytochrome b6 is tightly bound to the polypeptide. We exploited this property to define a pathway for the conversion of apo- to holocytochrome b6, and to identify mutants that are blocked at one step of this pathway. Chlamydomonas reinhardtii strains carrying substitutions in either one of the four histidines that coordinate the bh or bl hemes to the apoprotein were created. These mutations resulted in the appearance of distinct immunoreactive species of cytochrome b6, which allowed us to specifically identify cytochrome b6 with altered bh or bl ligation. In gabaculine-treated (i.e. heme-depleted) wild type and site-directed mutant strains, we established that (i) the single immunoreactive band, observed in strains carrying the bl site-directed mutations, corresponds to apocytochrome b6 and (ii) the additional band present in strains carrying bh site-directed mutations corresponds to a bl-heme-dependent intermediate in the formation of holocytochrome b6. Five nuclear mutants (ccb strains) that are defective in holocytochrome b6 formation display a phenotype that is indistinguishable from that of strains carrying site-directed bh ligand mutants. The defect is specific for cytochrome b6 assembly, because the ccb strains can synthesize other b cytochromes and all c-type cytochromes. The ccb strains, which define four nuclear loci (CCB1, CCB2, CCB3, and CCB4), provide the first evidence that a b-type cytochrome requires trans-acting factors for its heme association.

Cyclohexanecarboxylic Acids↗
PubMed1997

Ccs1, a nuclear gene required for the post-translational assembly of chloroplast c-type cytochromes.

Nuclear genes play important regulatory roles in the biogenesis of the photosynthetic apparatus of eukaryotic cells by encoding factors that control steps ranging from chloroplast gene transcription to post-translational processes. However, the identities of these genes and the mechanisms by which they govern these processes are largely unknown. By using glass bead-mediated transformation to generate insertional mutations in the nuclear genome of Chlamydomonas reinhardtii, we have generated four mutants that are defective in the accumulation of the cytochrome b6f complex. One of them, strain abf3, also fails to accumulate holocytochrome c6. We have isolated a gene, Ccs1, from a C. reinhardtii genomic library that complements both the cytochrome b6f and cytochrome c6 deficiencies in abf3. The predicted protein product displays significant identity with Ycf44 from the brown alga Odontella sinensis, the red alga Porphyra purpurea, and the cyanobacterium Synechocystis strain PCC 6803 (25-33% identity). In addition, we note limited sequence similarity with ResB of Bacillus subtilis and an open reading frame in a homologous operon in Mycobacterium leprae (11-12% identity). On the basis of the pleiotropic c-type cytochrome deficiency in the ccs1 mutant, the predicted plastid localization of the protein, and its relationship to candidate cytochrome biosynthesis proteins in Gram-positive bacteria, we conclude that Ccs1 encodes a protein that is required for chloroplast c-type holocytochrome formation.

Amino Acid Sequence↗
PubMed1996

Molecular genetic analysis of plastocyanin biosynthesis in Chlamydomonas reinhardtii.

Five plastocyanin-deficient mutants were identified from a population of UV-mutagenized Chlamydomonas reinhardtii cells. Genetic complementation experiments indicated that four mutants represented alleles at the PCY1 locus (pcy1-2, pcy1-3, pcy1-4, and pcy1-5). Sequence analysis confirmed that two strains, pcy1-2 and pcy1-3, carry a frameshift (-1) and a nonsense mutation, respectively, while strains pcy1-4 and pcy1-5 synthesize an extended protein as a result of read-through mutations at the stop codon. The C-terminal extension does not affect synthesis or processing of the pre-proteins, but the polypeptides are rapidly degraded after the second (lumenal) processing event. The frameshift mutation in pcy1-2 results in loss of Pcy1 mRNA, as noted previously for strain ac208 (pcy1-1), but the abundance of Pcy1 mRNA in strain pcy1-3, which carries a nonsense mutation at codon 26, is unaffected relative to wild-type cells. The decreased abundance of frameshifted Pcy1 mRNA is attributed to increased degradation rather than decreased synthesis, since the mRNAs can be stabilized by treatment of cells with cycloheximide or anisomycin. The fifth strain has a wild-type plastocyanin-encoding gene, but the strain accumulates apoplastocyanin at the expense of holoplastocyanin. We suggest that the mutation identifies a new locus (PCY2) whose function is required for normal holoplastocyanin accumulation. Like ac208 (pcy1-1), several of the new mutants were suppressed spontaneously owing to accumulation of cytochrome c6 (a functional substitute for plastocyanin). The suppressor mutation(s) displayed Mendelian inheritance and segregated independently from the PCY1 locus, which confirms that regulation of Cyc6 expression is not tightly linked to plastocyanin function.

Alleles↗
PubMed1996

The plastid-encoded ccsA gene is required for heme attachment to chloroplast c-type cytochromes.

A chloroplast gene, ycf5, which displays limited sequence identity to bacterial genes (ccl1/cycK) required for the biogenesis of c-type cytochromes, was tested for its function in chloroplast cytochrome biogenesis in Chlamydomonas reinhardtii. Targeted inactivation of the ycf5 gene results in a non-photosynthetic phenotype attributable to the absence of c-type cytochromes. The cloned ycf5 gene also complements the phototrophic growth deficiency in strain B6 of C. reinhardtii. B6 is unable to synthesize functional forms of cytochromes f and c6 owing to a chloroplast genome mutation that prevents heme attachment. The selected (phototrophic growth) as well as the unselected (holocytochrome c6 accumulation) phenotypes were restored in complemented strains. The complementing gene, renamed ccsA (for c-type cytochrome synthesis), is expressed in wild-type and B6 cells but is non-functional in B6 owing to an early frameshift mutation. Antibodies raised against the ccsA gene product recognize a 29-kDa protein in C. reinhardtii. The 29-kDa protein is absent in strain B6 but is restored in a spontaneous revertant (B6R) isolated from a culture of B6. Sequence analysis of the ccsA gene in strain B6R indicates that it is a true revertant. We conclude that the ccsA gene is expressed and that it encodes a protein required for heme attachment to c-type cytochromes.

Amino Acid Sequence↗
PubMed1996

Regulated copper uptake in Chlamydomonas reinhardtii in response to copper availability.

A saturable and temperature-dependent copper uptake pathway has been identified in Chlamydomonas reinhardtii. The uptake system has a high affinity for copper ions (Km approximately 0.2 microM) and is more active in cells that are adapted to copper deficiency than to cells grown in a medium containing physiological (submicromolar to micromolar) copper ion concentrations. The maximum velocity of copper uptake by copper-deficient cells (169 pmol h-1 10(6) cells-1 or 62 ng min-1 mg-1 chlorophyll) is up to 20-fold greater than that of fully copper-supplemented cells, and the Km (approximately 2 x 10(2) nM) is unaffected. Thus, the same uptake system appears to operate in both copper-replete and copper-deficient cells, but its expression or activity must be induced under copper-deficient conditions. A cupric reductase activity is also increased in copper-deficient compared with copper-sufficient cells. The physiological characteristics of the regulation of this cupric reductase are compatible with its involvement in the uptake pathway. Despite the operation of the uptake pathway under both copper-replete and copper-deficient conditions, C. reinhardtii cells maintained in fully copper-supplemented cells do not accumulate copper in excess of their metabolic need. These results provide evidence for a homeostatic mechanism for copper metabolism in C. reinhardtii.

Animals↗
PubMed1995

Degradation of plastocyanin in copper-deficient Chlamydomonas reinhardtii. Evidence for a protease-susceptible conformation of the apoprotein and regulated proteolysis.

In the green alga Chlamydomonas reinhardtii, the copper-dependent accumulation of plastocyanin is effected via the altered stability of the protein in copper-deficient versus copper-sufficient medium (t1/2) < 20 min versus several hours). To understand the mechanism of plastocyanin degradation in vivo, the purified apoprotein was characterized relative to the holoprotein with respect to conformation and protease susceptibility. Circular dichroism spectroscopy revealed that the apoprotein in solution did not display the characteristic secondary structure displayed by the native or reconstituted holoprotein. The apoprotein was also susceptible to digestion in vitro by chymotrypsin whereas the holoprotein was resistant. High ionic conditions, which stabilize the folded structure of apoplastocyanin, also inhibit its degradation by chymotrypsin. These results suggest that one explanation for plastocyanin degradation in copper-deficient cells in vivo might be the increased susceptibility of the apo form to a lumenal protease. Since apoplastocyanin is a normal biosynthetic intermediate for the formation of holoplastocyanin, the increased susceptibility of apoplastocyanin to proteolysis implies that degradative and biosynthetic activities would compete for the same substrate. However, characterization of an apoplastocyanin-accumulating mutant suggests that a plastocyanin-degrading protease is active only in copper-deficient cells. Thus, apoplastocyanin is rapidly degraded in copper-deficient cells, whereas its major fate in copper-supplemented cells is holoplastocyanin formation.

Animals↗
PubMed1995

The structure of chloroplast cytochrome c6 at 1.9 A resolution: evidence for functional oligomerization.

The molecular structure of cytochrome c6 from the green alga Chlamydomonas reinhardtii has been determined from two crystal forms and refined to 1.9 A resolution. The two crystal forms are likely the result of different levels of post-translational modification of the protein. This is the first report of a high-resolution structure of a chloroplast-derived class I c-type cytochrome. The overall fold is similar to that of other class I c-type cytochromes, consisting of a series of alpha-helices and turns that envelop the heme prosthetic group. There is also a short two-stranded anti-parallel beta-sheet in the vicinity of the methionine axial ligand to the heme; this region of the molecule is formed by the most highly conserved residues in c6-type cytochromes. Although class I c-type cytochromes are assumed to function as monomers, both crystal forms of cytochrome c6 exhibit oligomerization about the heme crevice that is, in part, mediated by the short anti-parallel beta-sheet. The functional significance of this oligomerization is supported by the appearance of similar interfaces in other electron transfer couples, HPLC and light-scattering data, and is furthermore consistent with kinetic data on electron transfer reactions of c6-type cytochromes.

Amino Acid Sequence↗
PubMed1995

Coordinate expression of coproporphyrinogen oxidase and cytochrome c6 in the green alga Chlamydomonas reinhardtii in response to changes in copper availability.

To maintain photosynthetic competence under copper-deficient conditions, the green alga Chlamydomonas reinhardtii substitutes a heme protein (cytochrome c6) for an otherwise essential copper protein, viz. plastocyanin. Here, we report that the gene encoding coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway, is coordinately expressed with cytochrome c6 in response to changes in copper availability. We have purified coproporphyrinogen oxidase from copper-deficient C.reinhardtii cells, and have cloned a cDNA fragment which encodes it. Northern hybridization analysis confirmed that the protein is nuclear-encoded and that, like cytochrome c6, its expression is regulated by copper at the level of mRNA accumulation. The copper-responsive expression of coproporphyrinogen oxidase parallels cytochrome c6 expression exactly. Specifically, the copper-sensing range and metal selectivity of the regulatory components, as well as the time course of the responses, are identical. Hence, we propose that the expression of these two proteins is controlled by the same metalloregulatory mechanism. Our findings represent a novel metalloregulatory response in which the synthesis of one redox cofactor (heme) is controlled by the availability of another (Cu).

Amino Acid Sequence↗
PubMed1995

Biosynthesis of cytochrome f in Chlamydomonas reinhardtii: analysis of the pathway in gabaculine-treated cells and in the heme attachment mutant B6.

Chlamydomonas reinhardtii uses two c-type cytochromes for photosynthetic electron transfer: the thylakoid membrane-bound cytochrome f of the cytochrome b6f complex and the soluble cytochrome c6. Previously, a class of photosynthesis-minus, acetate-requiring mutants was identified which were deficient in both c-type cytochromes, and biochemical analyses of cytochrome c6 biosynthesis in these strains indicated that they were each blocked at the step of heme attachment to apocytochrome c6. In order to demonstrate that the deficiency in cytochrome f results from the same biochemical and genetic defect, cytochrome f biosynthesis was examined in the B6 mutant (a representative of this phenotypic class) and in spontaneous suppressor strains derived from B6. Pulse-radiolabeling experiments show that B6 synthesizes a form of cytochrome f that is rapidly degraded in vivo. This polypeptide is membrane associated and migrates with an electrophoretic mobility identical to that of standard apocytochrome f produced in vitro but slightly greater than that of standard holocytochrome f produced in vivo by wild-type cells. These findings suggest that the B6 strain is unable to convert apocytochrome f to holocytochrome f and that apocytochrome f is unstable in vivo. In the suppressed strains, accumulation of both holocytochrome f and holocytochrome c6 is restored. One suppressor mutation (strain B6R) displays uniparental inheritance whereas another (B6T3) displays Mendelian inheritance. In both cases, the three phenotypes, photosynthesis-plus, b6f+ and cyt c6+ co-segregate in genetic crosses. This study therefore confirms that the dual cyt b6f-/cytc6- deficiency in B6 results from a single mutation that affects a step in holocytochrome formation that is common to the biosynthetic pathways of both plastidic c-type cytochromes. The study also confirms that pre-apocytochrome f synthesis, processing and association with the membrane is not dependent on heme attachment. Synthesis of cytochrome f does, however, appear to be dependent on heme availability. In cells depleted of tetrapyrrole pathway intermediates by gabaculine treatment, cytochrome f synthesis was significantly reduced. Since gabaculine treatment did not affect the stability of cytochrome f nor the accumulation of cytochrome f-encoding transcripts, the reduction is attributed to post-transcriptional regulation of preapocytochrome f synthesis via a pathway that is sensitive to the availability of heme or a tetrapyrrole pathway intermediate.

Animals↗
PubMed1995

Two copper-responsive elements associated with the Chlamydomonas Cyc6 gene function as targets for transcriptional activators.

In Chlamydomonas reinhardtii, cytochrome c6 (cyt c6) is synthesized only under conditions of copper deficiency when plastocyanin cannot be synthesized. In previous work, the copper-responsive regulation of cyt c6 synthesis was demonstrated to occur by control of transcription, with no contribution from post-transcriptional processes. To understand the mechanism underlying its regulation, the genomic DNA encoding cyt c6 (Cyc6) was analyzed for the presence of copper-responsive elements. Sequences lying between positions -127 and -7 with respect to the start site of transcription were found to be sufficient to confer copper-responsive expression on either a promoterless or a minimal beta-tubulin promoter-driven (arylsulfatase-encoding) reporter gene. Analysis of this 120-bp fragment indicated that copper-responsive elements lie in two distinct regions (between -110 to -56 and -127 to -109). ATG fusions between copper-insensitive promoters and the coding plus 3' untranslated region of the Cyc6 gene resulted in the accumulation of cyt c6 in copper-supplemented medium; this confirms earlier studies indicating a lack of post-transcriptional control in this copper-responsive pathway. In the context of a constitutive promoter (derived from the beta-tubulin gene), each region was found to function as an activator of transcription in copper-deficient cells, and the metal specificity of the response of reporter genes containing either one or both regions was identical to that of the endogenous Cyc6 gene. The copper-responsive synthesis of cyt c6 is thus attributed to these two 5' upstream sequences.

Animals↗
PubMed1994

Role of heme in the biosynthesis of cytochrome c6.

Cytochrome c6, a nuclear-encoded protein, is synthesized in the cytoplasm in a precursor form (pre-apocytochrome c6). Time course radiolabeling experiments support the model that the pre-protein is the substrate for a lumen-targeting post-translational pathway which includes two proteolytic cleavage events and the covalent attachment of heme to cysteinyl thiols on the polypeptide. Cell fractionation studies indicate that the fully processed protein fractionates with other soluble proteins, whereas the precursor and intermediate forms appear to be membrane-associated. To determine whether either of the processing steps is influenced by heme attachment, the biosynthetic pathway was examined 1) in a Chlamydomonas reinhardtii mutant (B6) that is defective in the heme attachment step and 2) in wild-type cells treated with gabaculine, an inhibitor of heme synthesis. In vivo radiolabeling experiments with the B6 mutant showed that a defect in heme attachment affects neither the synthesis of the pre-apoprotein nor its processing to the mature apoform to any significant extent, supporting the notion that heme attachment and processing are not obligatorily coupled in this pathway. A similar conclusion is reached from examination of cytochrome c6 synthesis in gabaculine-treated cells where inhibition of heme attachment did not prevent either of the two processing steps. The fully processed apoprotein is a suitable substrate for heme attachment in vivo, since the apoprotein was indeed converted to holoprotein in gabaculine-treated cells, albeit more slowly than in untreated cells. Despite the lack of effect of gabaculine treatment on the accumulation of cytochrome c6-encoding messages, the amount of holocytochrome c6 precursors synthesized during a brief labeling period is 4-7-fold less than in untreated cells, suggesting that synthesis of the polypeptide may be coupled to heme availability by a control mechanism operating at the translational level.

Animals↗
PubMed1994

Plastocyanin: structural and functional analysis.

Plastocyanin is one of the best characterized of the photosynthetic electron transfer proteins. Since the determination of the structure of poplar plastocyanin in 1978, the structure of algal (Scenedesmus, Enteromorpha, Chlamydomonas) and plant (French bean) plastocyanins has been determined either by crystallographic or NMR methods, and the poplar structure has been refined to 1.33 A resolution. Despite the sequence divergence among plastocyanins of algae and vascular plants (e.g., 62% sequence identity between the Chlamydomonas and poplar proteins), the three-dimensional structures are remarkably conserved (e.g., 0.76 A rms deviation in the C alpha positions between the Chlamydomonas and poplar proteins). Structural features include a distorted tetrahedral copper binding site at one end of an eight-stranded antiparallel beta-barrel, a pronounced negative patch, and a flat hydrophobic surface. The copper site is optimized for its electron transfer function, and the negative and hydrophobic patches are proposed to be involved in recognition of physiological reaction partners. Chemical modification, cross-linking, and site-directed mutagenesis experiments have confirmed the importance of the negative and hydrophobic patches in binding interactions with cytochrome f and Photosystem I, and validated the model of two functionally significant electron transfer paths in plastocyanin. One putative electron transfer path is relatively short (approximately 4 A) and involves the solvent-exposed copper ligand His-87 in the hydrophobic patch, while the other is more lengthy (approximately 12-15 A) and involves the nearly conserved residue Tyr-83 in the negative patch.

Amino Acid Sequence↗
PubMed1993

The 1.5-A crystal structure of plastocyanin from the green alga Chlamydomonas reinhardtii.

The crystal structure of plastocyanin from the green alga Chlamydomonas reinhardtii has been determined at 1.5-A resolution with a crystallographic R factor of 16.8%. Plastocyanin is a small (98 amino acids), blue copper-binding protein that catalyzes the transfer of electrons in oxygenic photosynthesis from cytochrome f in the quinol oxidase complex to P700+ in photosystem I. Chlamydomonas reinhardtii plastocyanin is an eight-stranded, antiparallel beta-barrel with a single copper atom coordinated in quasitetrahedral geometry by two imidazole nitrogens (from His-37 and His-87), a cysteine sulfur (from Cys-84), and a methionine sulfur (from Met-92). The molecule contains a region of negative charge surrounding Tyr-83 (the putative distant site of electron transfer) and an exclusively hydrophobic region surrounding His-87; these regions are thought to be involved in the recognition of reaction partners for the purpose of directing electron transfer. Chlamydomonas reinhardtii plastocyanin is similar to the other plastocyanins of known structure, particularly the green algal plastocyanins from Enteromorpha prolifera and Scenedesmus obliquus. A potential "through-bond" path of electron transfer has been identified in the protein that involves the side chain of Tyr-83, the main-chain atoms between residues 83 and 84, the side chain of Cys-84, the copper atom, and the side chain of His-87.

Amino Acid Sequence↗
PubMed1993

The plastocyanin-deficient phenotype of Chlamydomonas reinhardtii Ac-208 results from a frame-shift mutation in the nuclear gene encoding preapoplastocyanin.

Ac-208 is a plastocyanin-deficient mutant of Chlamydomonas reinhardtii that contains only 2-3% of the wild-type level of plastocyanin-encoding mRNA and no detectable plastocyanin. Sequence analysis of the ac-208 plastocyanin-encoding gene reveals a single nucleotide insertion in the first exon compared with the wild-type gene; this alters the reading frame and results in a premature nonsense codon. We have introduced the genomic sequence encoding plastocyanin from a wild-type strain into ac-208 by cotransformation with a selectable marker encoding nitrate reductase. Of 22 nit+ transformants characterized, nine contained additional plastocyanin-encoding sequences (compared with untransformed cells) and each of these nine transformants was found to accumulate the protein. Transformants that do not contain newly introduced plastocyanin sequences retain the plastocyanin-deficient phenotype. The introduced plastocyanin-encoding sequences are stable during mitotic growth in liquid culture over a period of several months, as is expression from the introduced sequences. We suggest that the decreased steady state level of plastocyanin-encoding messages is a consequence of the frame-shift mutation in the structural gene. The ability to complement ac-208 with plastocyanin-encoding sequences will allow the introduction and analysis of in vitro mutagenized plastocyanin sequences in vivo in transgenic C. reinhardtii cells.

Amino Acid Sequence↗
PubMed1993

Maturation of thylakoid lumen proteins proceeds post-translationally through an intermediate in vivo.

Many thylakoid lumen proteins are synthesized outside the chloroplast as larger molecular weight precursors and then processed to their mature size during transport to the lumenal space. We have examined the post-translational processing of thylakoid lumen proteins in vivo by pulse-radiolabeling experiments with Chlamydomonas reinhardtii. Antibodies against the lumenal protein cytochrome c6 specifically immunoprecipitated three polypeptides from extracts of briefly pulse-radiolabeled cells. The molecular weights and kinetics of synthesis and turnover indicate that these three polypeptides are (i) the full-length cytochrome c6 precursor, (ii) a partially processed precursor (intermediate), and (iii) the completely processed mature protein. Identification of analogous forms of two other lumenal proteins, plastocyanin and the oxygen evolving enhancer 1 protein, indicates that the maturation of thylakoid lumen proteins occurs post-translationally in vivo and that the partially processed intermediate is a general feature of the pathway. The intermediate form of cytochrome c6 accumulated to a greater extent in cells incubated at 10 degrees C, relative to cells incubated at 22 degrees C, concomitantly with a decrease in the accumulation of the mature protein. The intermediate accumulating at 10 degrees C is quantitatively converted to the mature protein upon incubation at higher temperature, thus demonstrating a precursor-product relationship between the intermediate and mature forms of cytochrome c6. Our results prove the model [Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K. & Weisbeek, P. (1986) Cell 46, 365-375] that precursors of lumenal proteins are post-translationally converted to their mature forms in two steps through a distinct intermediate.

Algal Proteins↗