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First published online January 12, 2007; 10.1105/tpc.106.042739

The Plant Cell 19:256-269 (2007)
© 2007 American Society of Plant Biologists

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Chlamydomonas reinhardtii Has Multiple Prolyl 4-Hydroxylases, One of Which Is Essential for Proper Cell Wall Assembly[W]

Katriina Keskiahoa, Reija Hietaa, Raija Sormunenb and Johanna Myllyharjua,1

a Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu, FIN-90014 Oulu, Finland
b Biocenter Oulu and Department of Pathology, University of Oulu, FIN-90014 Oulu, Finland

1 To whom correspondence should be addressed. E-mail johanna.myllyharju{at}oulu.fi; fax 358-8-537 5811.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
Prolyl 4-hydroxylases (P4Hs) catalyze formation of 4-hydroxyproline (4Hyp), which is found in many plant glycoproteins. We cloned and characterized Cr-P4H-1, one of 10 P4H-like Chlamydomonas reinhardtii polypeptides. Recombinant Cr-P4H-1 is a soluble 29-kD monomer that effectively hydroxylated in vitro both poly(L-Pro) and synthetic peptides representing Pro-rich motifs found in the Chlamydomonas cell wall Hyp-rich glycoprotein (HRGP) GP1. Similar Pro-rich repeats that are likely to be Cr-P4H-1 substrates are also present in the cell wall HRGP GP2 and probably GP3. Suppression of the gene encoding Cr-P4H-1 by RNA interference led to a defective cell wall consisting of a loose network of fibrils resembling the inner and outer W1 and W7 layers of the wild-type wall, while the layers forming the dense central triplet were absent. The lack of Cr-P4H-1 most probably affected 4Hyp content of the major HRPGs of the central triplet, GP1, GP2, and GP3. The reduced 4Hyp levels in these HRGPs can also be expected to affect their glycosylation and, thus, the interactive properties and stabilities of their fibrous shafts. Interestingly, our RNA interference data indicate that the nine other Chlamydomonas P4H-like polypeptides could not fully compensate for the lack of Cr-P4H-1 activity and are therefore likely to have different substrate specificities and functions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
4-Hydroxyproline (4Hyp) is found in many Hyp-rich plant glycoproteins (HRGPs) that are the major structural components of cell walls (for reviews, see Cassab, 1998Go; Kieliszewski and Shpak, 2001Go). The HRGP family contains three major subgroups: repetitive Pro-rich proteins, extensins, and arabinogalactan proteins (Cassab, 1998Go; Kieliszewski and Shpak, 2001Go). The cell walls in green algae are almost entirely built up from extensin-like HRGPs, the Chlamydomonas reinhardtii cell wall containing 25 to 30 different HRGPs (Adair and Snell, 1990Go; Sumper and Hallmann, 1998Go). In addition, sexual adhesion of C. reinhardtii gametes during mating occurs via agglutinins that are also HRGPs (Ferris et al., 2005Go). Three classes of Pro-rich motifs that consist of contiguous Pro residues, Pro residues alternating with some other amino acid, mostly Ser, and -Pro-Pro-Ser-Pro-X- repeats have been identified in algal HRGPs, with many of the Pro residues being subsequently 4-hydroxylated (Adair and Snell, 1990Go; Sumper and Hallmann, 1998Go; Ferris et al., 2001Go).

Most of the 4Hyp in animal proteins is found in collagens and >20 additional proteins with collagen-like sequences. 4Hyp residues play a critical role in all collagens, as they are essential for the formation of stable collagen triple helices at body temperature (for reviews, see Kivirikko and Pihlajaniemi, 1998Go; Myllyharju, 2003Go; Myllyharju and Kivirikko, 2004Go).

The formation of 4Hyp in all these proteins is catalyzed by prolyl 4-hydroxylases (P4Hs), which act on Pro residues in peptide linkages and require Fe2+, 2-oxoglutarate, O2, and ascorbate (Kivirikko and Pihlajaniemi, 1998Go; Myllyharju, 2003Go). The well-characterized vertebrate type I, II, and III collagen P4Hs (C-P4Hs) reside within the lumen of the endoplasmic reticulum and are {alpha}2ß2 tetramers in which the catalytic {alpha} subunits have three isoforms: {alpha}(I), {alpha}(II), and {alpha}(III) (Helaakoski et al., 1989Go; Annunen et al., 1997Go; Kukkola et al., 2003Go; Van Den Diepstraten et al., 2003Go). Animal C-P4Hs have also been identified and characterized from nematodes (Veijola et al., 1994Go; Friedman et al., 2000Go; Winter and Page, 2000Go; Merriweather et al., 2001Go; Myllyharju et al., 2002Go; Riihimaa et al., 2002Go; Winter et al., 2003Go) and Drosophila melanogaster (Annunen et al., 1999Go; Abrams and Andrew, 2002Go). A second family of animal P4Hs has a cytoplasmic and nuclear location. It consists of three isoenzymes with no ß subunit that play a central role in the oxygen-dependent regulation of the hypoxia-inducible transcription factor HIF (Bruick and McKnight, 2001Go; Epstein et al., 2001Go; Ivan et al., 2002Go). These HIF-P4Hs are effective oxygen sensors due to their high Km values for O2 (Hirsilä et al., 2003Go).

Plant P4Hs have been partially purified and characterized from many higher plant sources (for a review, see Kivirikko et al., 1992Go) and the green algae C. reinhardtii and Volvox carteri (Kaska et al., 1987Go, 1988Go), but the only plant P4Hs cloned and characterized so far are two monomeric enzymes, At-P4Hs 1 and 2, from Arabidopsis thaliana and one from Nicotiana tabacum (Hieta and Myllyharju, 2002Go; Tiainen et al., 2005Go; Yuasa et al., 2005Go). Both recombinant At-P4Hs effectively hydroxylate in vitro both poly(L-Pro) and many synthetic peptides corresponding to Pro-rich repeats present in plant glycoproteins but have distinct differences in their substrate specificities (Hieta and Myllyharju, 2002Go; Tiainen et al., 2005Go). A monomeric P4H has also been cloned and characterized from the Paramecium bursaria Chlorella virus-1 (PBCV-1) and has been found to share some of the substrate binding properties of the At-P4Hs (Eriksson et al., 1999Go).

The overall amino acid sequence identities between the {alpha} subunits of animal C-P4Hs and the HIF-P4Hs and plant P4Hs are relatively low, but the catalytically critical residues are conserved (Myllyharju, 2003Go). We report here that the C. reinhardtii genome contains 10 genes encoding P4H-like polypeptides. One of the cDNAs, encoding a polypeptide named Cr-P4H-1, was cloned and expressed as a soluble 29-kD recombinant monomer with a unique substrate specificity as compared with those of the P4Hs from higher plants and animal sources. The recombinant Cr-P4H-1 efficiently hydroxylated synthetic peptides representing Pro-rich motifs of the C. reinhardtii cell wall proteins GP1 and GP2. Cr-P4H-1 was found to be essential for proper cell wall assembly in vivo, as suppression of its gene by RNA interference (RNAi) led to inability of the treated cells to regenerate their walls.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 METHODS
 REFERENCES
 
The C. reinhardtii Genome Encodes 10 Putative P4H Polypeptides
Although monomeric P4Hs have been partially purified from C. reinhardtii and V. carteri (Kaska et al., 1987Go, 1988Go), no sequence information on the algal enzymes has been available. A sequence homology search in GeneBank with the catalytic C-terminal region of the human C-P4H {alpha}(I) subunit (Helaakoski et al., 1989Go) indicated the presence of overlapping C. reinhardtii EST clones derived from a single open reading frame that encodes a P4H-like polypeptide of 253 residues (Figure 1A ). The polypeptide, named here Cr-P4H-1, was predicted to have a 16-residue N-terminal signal peptide. The sequence of the processed Cr-P4H-1 polypeptide is 26% identical to that of the catalytic C-terminal region of the human C-P4H {alpha}(I) subunit, the identity with the two low molecular weight P4Hs from Arabidopsis (Hieta and Myllyharju, 2002Go; Tiainen et al., 2005Go) being 40% and that with the PBCV-1 P4H (Eriksson et al., 1999Go) 30% (Figure 1A), whereas the identity with the C-terminal regions of the three human HIF-P4Hs is only 11 to 14% (data not shown). The two His residues and one Asp that bind the Fe2+ atom and the Lys that binds the C-5 carboxyl group of the 2-oxoglutarate at the catalytic site in the C-P4Hs, At-P4Hs, and PBCV-1 P4H (Lamberg et al., 1995Go; Myllyharju and Kivirikko, 1997Go; Eriksson et al., 1999Go; Hieta and Myllyharju, 2002Go; Tiainen et al., 2005Go) are all conserved in the Cr-P4H-1 sequence (Figure 1A). The fifth critical residue, a His in most C-P4H sequences and At-P4H-2, which is probably involved in the binding of the C-1 carboxyl group of 2-oxoglutarate to the Fe2+ atom and the decarboxylation of this cosubstrate (Myllyharju and Kivirikko, 1997Go), is also conserved in Cr-P4H-1, while in At-P4H-1 and PBCV-1 P4H, this residue is replaced by an Arg (Eriksson et al., 1999Go; Hieta and Myllyharju, 2002Go). The Cr-P4H-1 polypeptide contains three Cys residues, which are all conserved in At-P4H-1 (Figure 1A).


Figure 1
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  		Figure 1. Amino Acid Sequence Comparisons of Cr-P4H-1.

Alignment of the amino acid residues of Cr-P4H-1A (A) with those of the Arabidopsis P4H-1 (At-P4H-1), the P. bursaria Chlorella virus-1 P4H (PBCV-1 P4H), and the C-terminal region of the human C-P4H {alpha}(I) subunit [Hs {alpha}(I)] and the Cr-P4H-1 (B) with those of the other putative C. reinhardtii P4Hs. The C-terminal extension present only in the variant Cr-P4H-1B is boxed in (B). The putative C. reinhardtii P4Hs are indicated in (B) by their annotation numbers in the C. reinhardtii genome draft release, version 2.0, of the Department of Energy Joint Genome Institute. The catalytically critical residues are indicated by asterisks. Identical amino acids are shown with black backgrounds.

 
No other P4H-like C. reinhardtii sequences were found in GenBank. However, analysis of the annotations in the C. reinhardtii genome draft release, version 2.0, of the Department of Energy Joint Genome Institute revealed the presence of at least nine other open reading frames encoding putative P4H polypeptides (Figure 1B). In addition, the annotation C_110025 indicated that there exists an alternatively spliced form of Cr-P4H-1 in which the last exon encoding the last three amino acids before the stop codon is replaced by an exon encoding a Cys-rich stretch of 47 residues. The sequence of this C-terminal extension is homologous to ShK, a 35-residue polypeptide potassium channel toxin from the sea anemone Stichodactyla helianthus (Dauplais et al., 1997Go; Pan et al., 1998Go), a similar homologous region having also been found previously in At-P4H-2 (Tiainen et al., 2005Go). The longer splice variant will be referred to below as Cr-P4H-1B and the shorter one as Cr-P4H-1A. An EST database search indicated that both variants are expressed in C. reinhardtii cells.

One of the other annotated sequences, C_360012, shows 41% amino acid sequence identity to Cr-P4H-1A and is also of the same length (Figure 1B). Eight of the sequences, namely C_390100, C_150026, C_280045, C_1000010, C_2500005, C_50128, C_130209, and C_800043, are of variable lengths and, like Cr-P4H-1B, also contain the C-terminal ShK toxin-like sequences (Figure 1B). EST sequences could be found for all putative P4H isoenzymes, except C_130209. Prediction of signal peptide cleavage sites indicated that all but three (C_360012, C_130209, and C_800043) of the isoforms have cleavable N-terminal signal peptides. The catalytically critical residues are conserved in all the sequences, except that the second iron binding His (corresponding to His-227 in Cr-P4H1) is replaced by a Glu in C_800043, and the fifth critical residue (His-245 in Cr-P4H1) is replaced by an Arg in C_2500005, as also in At-P4H-1 and PBCV-1 P4H.

Phylogenetic analysis showed that Cr-P4H-1 is evolutionarily closest to At-P4H-1, these polypeptides forming a clade with At-P4H-2. These algal and plant P4Hs are also closely related to the other low molecular weight P4Hs, namely the viral PBCV-1 P4H and the shortest Caenorhabditis elegans C-P4H, PHY-3 (Figure 2 ). Furthermore, when compared with the longer, ~500-residue, catalytic {alpha}-subunits of the vertebrate and C. elegans C-P4Hs, the algal and plant P4Hs are more closely related to the human, mouse, and rat {alpha}(III) subunit isoforms than to the {alpha}(I) and {alpha}(II) subunits or to the C. elegans {alpha}-subunits PHY-1 and PHY-2 (Figure 2). The animal HIF-P4Hs were not included in this analysis due to their low sequence identity with the other P4Hs.


Figure 2
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  		Figure 2. Phylogenetic Tree of Cr-P4H-1 and Other P4Hs.

Phylogenetic relationships between the amino acid sequences of Cr-P4H-1A and the C-P4H {alpha} subunits from human [Hs {alpha}(I), {alpha}(II), and {alpha}(III)], mouse [Mm {alpha}(I), {alpha}(II), and {alpha}(III)], rat [Rn {alpha}(I) and {alpha}(III)], chicken [Gg {alpha}(I)], C. elegans (Ce PHY-1, PHY-2, and PHY-3), Brugia malayi (Bm PHY-1), Onchocerca volvulus (Ov PHY-1), D. melanogaster [Dm {alpha}(I)], and P4Hs from Arabidopsis (At-P4H-1 and At-P4H-2) and PBCV-1 P4H. The neighbor-joining tree was constructed using MEGA, version 2.1 (Kumar et al. 2001Go). Bootstrap values determined from 1000 replications are shown at each node. Bar = 10% divergence.

 
Expression and Analysis of Recombinant Cr-P4H-1A and Cr-P4H-1B
A cDNA encoding the full-length Cr-P4H-1A was amplified from a C. reinhardtii cDNA library, cloned into the baculovirus vector pVL1392, and used to generate a recombinant virus. Insect cells were harvested 72 h after infection with the virus, homogenized in a buffer containing 0.1% Triton X-100, and centrifuged. The remaining pellet was further solubilized in 1% SDS, and the samples were analyzed by SDS-PAGE and Coomassie blue staining. Most of the recombinant Cr-P4H-1A remained in the insoluble fraction (Figure 3A , lane 2), but a distinct protein band of the correct size was also seen in the soluble fraction (Figure 3A, lane 1). Cr-P4H-1A was purified from the Triton X-100 soluble fraction by gel filtration on a HiPrep Sephacryl S100 HR column, and the fractions were analyzed by SDS-PAGE. Surprisingly, essentially homogeneous Cr-P4H-1A was obtained by this single-step gel filtration procedure (Figure 3A, lane 3). Gel filtration experiments using a calibrated SuperDex 75 HR column showed that the pure enzyme was eluted in fractions that corresponded to a molecular weight of ~35,000 D (data not shown). This indicates that the recombinant Cr-P4H-1A is a monomer, as the calculated molecular weight of the recombinant His-tagged Cr-P4H-1A without the signal peptide is 28,315 D.


Figure 3
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  		Figure 3. Expression of Recombinant Cr-P4H-1A and Cr-P4H-1B Polypeptides in Insect Cells or in E. coli and Their Purification.

The recombinant Cr-P4H-1A was expressed in insect cells (A) and in E. coli (B), and Cr-P4H-1B was expressed in E. coli (C). Samples of the soluble (lane 1) and insoluble (lane 2) fractions of the cell lysates were analyzed by SDS-PAGE under reducing conditions. The Cr-P4H-1A was purified from insect cells by gel filtration (lane 3 in [A]), and Cr-P4H-1A and Cr-P4H-1B were purified from E. coli using a Ni-NTA Sepharose column (lane 3 in [B] and [C]).

 
A cDNA encoding the Cr-P4H-1A residues Ala-17–His-253 (i.e., lacking the predicted signal sequence) was cloned into the pET15b Escherichia coli expression vector with an N-terminal His6 tag and transformed into the E. coli Origami (DE3) strain. The cells were harvested after overnight induction at 20°C and sonicated, and the soluble and insoluble fractions were analyzed by SDS-PAGE. More than half of the recombinant Cr-P4H-1A was found in the soluble fraction (Figure 3B, compare lanes 1 and 2) and could be purified essentially to homogeneity using a Ni-NTA column and gel filtration (Figure 3B, lane 3). A cDNA encoding the Cr-P4H-1B residues Ala-17–Thr-297 was cloned, expressed, and similarly purified essentially to homogeneity (Figure 3C, lane 3).

The thermal stability of the purified recombinant Cr-P4H-1A expressed in E. coli was studied by means of circular dichroism (CD) spectroscopy. The polypeptide displayed a cooperative transition of the folded state to an unfolded state during a thermal scan from 20 to 80°C, the melting temperature being ~58°C (Figure 4 ). These results indicate that the recombinant polypeptide has a fairly compact, stable fold.


Figure 4
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  		Figure 4. CD Analysis of the Temperature Stability of the Recombinant Cr-P4H-1A Polypeptide.

Mean residue ellipticity at 222 nm as a function of temperature (T) measured during denaturation from 20 to 90°C. mrw, mean residue weight.

 
The P4H activity of the purified Cr-P4H-1A from the insect cell and E. coli expressions and the Cr-P4H-1B from the E. coli expressions was analyzed by a method based on measurement of the hydroxylation-coupled decarboxylation of 2-oxo[1-14C]glutarate. The recombinant Cr-P4H-1A from both hosts and Cr-P4H-1B showed P4H activity when poly(L-Pro), Mr 5000 or 30,000, was used as a substrate. The specific activities of Cr-P4H-1A and Cr-P4H-1B were ~300 mol/mol enzyme/min in the presence of 60 µM poly(L-Pro), Mr 30,000, under standard reaction conditions. This value is similar to that obtained for the recombinant human type I C-P4H when the collagen-like peptide (Pro-Pro-Gly)10 was used as a substrate (Vuori et al., 1992Go). As the specific activities of Cr-P4H-1A and Cr-P4H-1B were identical, Cr-P4H-1A was selected for subsequent determination of Km values for the reaction cosubstrates and various peptide substrates and enzyme inhibition constant (Ki) values for certain inhibitors. The Km for Fe2+ was 30 µM, being approximately twofold to sixfold higher than those for the recombinant At-P4Hs 1 and 2 and 75-fold higher than that for the recombinant PBCV-1 P4H (Table 1 ). The Km for 2-oxoglutarate, 250 µM, was 1.5- to 2-fold higher than the corresponding values for the At-P4Hs 1 and 2 and >10-fold higher than that for the PBCV-1 P4H (Table 1), while the Km for ascorbate, 20 µM, was 15-fold lower than the corresponding values for the At-P4Hs 1 and 2 and the PBCV-1 P4H (Table 1).


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  		Table 1. Km Values of C. reinhardtii P4H-1, Arabidopsis P4Hs 1 and 2, and PBCV-1 P4H for Cosubstrates and Poly(L-Pro) and Ki Values for Certain Inhibitors

 
The 2-oxoglutarate analogs pyridine-2,4-dicarboxylate and pyridine-2,5-dicarboxylate, which are effective competitive inhibitors of vertebrate C-P4Hs, were found to inhibit Cr-P4H-1, but only at relatively high concentrations, the Ki values being 70 and 100 µM, respectively. These values are 35-fold and 125-fold higher than the corresponding values for human C-P4H-I, and 7-fold and 1.3-fold higher than those for At-P4H-2. Zn2+ was found to be quite an effective inhibitor with respect to Fe2+, the Ki value being 10 µM, which is nevertheless markedly higher than the Ki of 0.6 µM determined for human C-P4H-I (Kivirikko et al., 1992Go).

Recombinant Cr-P4H-1 Hydroxylates Poly(L-Pro), C. reinhardtii HRGP Motifs, and Collagen-Like Peptides
The Km values of the recombinant Cr-P4H-1A for poly(L-Pro) peptides of two molecular weights, Mr 5000 and 30,000, were 140 and 25 µM, respectively (Table 1), being markedly higher than those of At-P4H-1 and also higher than those of At-P4H-2, but lower than the values of PBCV-1 P4H (Table 1). The Km of Cr-P4H-1 for poly(L-Pro) decreased with increasing chain length of the peptide (Table 1), which is a typical finding also in the case of animal C-P4Hs (Kivirikko and Myllylä, 1982Go; Kivirikko and Pihlajaniemi, 1998Go; Myllyharju, 2003Go). The recombinant Cr-P4H-1 differed from the animal C-P4Hs in that the Km for each single Pro in the substrate did not decrease with increasing substrate length (Table 1) but remained essentially unchanged, as has also been observed with the partially purified C. reinhardtii enzyme (Kaska et al., 1987Go).

The C. reinhardtii genome contains gene families encoding HRGPs that have at least three kinds of Pro-rich motifs subjected to posttranslational hydroxylation (Ferris et al., 2001Go). The motifs may be strings of contiguous Pro residues or Pro residues alternating with other amino acids, most often Ser, and some HRGPs have long, regular repeats of the -Pro-Pro-Ser-Pro-X- motif. The synthetic peptides (Pro-Ser-Pro-Ser-Pro-Ser-Pro-Ser-Pro-Ser-Pro-Ser-Pro-Ser-Pro-Ser-Pro-Ser-Pro-Ser-Pro-Ser-Pro-Ser-Pro-Ser-Pro-Ile-Pro-Ser-Pro-Ser-Pro-Lys-Pro-Ser-Pro-Ser-Pro), representing the (Ser-Pro)19 repeat in domain 3 of the C. reinhardtii cell wall GP1 protein, and (Ser-Pro-Ala/Glu/Lys-Pro-Pro)5, representing the -Pro-Pro-Ser-Pro-X- motif in domain 2 of GP1, the C. reinhardtii sexual agglutinins Sag1 and Sad1, and a related protein A2 of unknown function (Ferris et al., 2001Go, 2005Go) all served as substrates for Cr-P4H-1A with essentially identical Km values, ~100 µM, which were also similar to that for poly(L-Pro), Mr 5000 (Table 2 ). The highest Vmax was obtained with the (Ser-Pro)19 peptide, this value being identical to that recorded with poly(L-Pro), Mr 5,000, and slightly lower than that with poly(L-Pro), Mr 30,000 (Table 2). Although the Km values for all the (Pro-Pro-Ser-Pro-X)5 peptides were 100 µM, their Vmax varied from 25 to 65% of that obtained with poly(L-Pro), Mr 30,000, the Vmax of (Ser-Pro-Ala-Pro-Pro)5 being the highest (Table 2). The lowest Vmax was obtained with (Ser-Pro-Lys-Pro-Pro)5, which may reflect the suggestion that a Pro following a Lys residue is not hydroxylated in higher plants (Kieliszewski and Lamport, 1994Go). This same peptide also corresponds to sequences encoded in the PBCV-1 genome and is a good substrate for the recombinant PBCV-1 P4H, with a Km of 20 µM (Eriksson et al., 1999Go).


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  		Table 2. Km Values of Recombinant C. reinhardtii P4H-1 for Synthetic Pro-Rich Peptides

 
The Cr-P4H-1A enzyme also hydroxylated a denatured collagen-like (Pro-Pro-Gly)10 peptide, but very inefficiently, with a Km of >1500 µM, 25-fold higher than that of At-P4H-1 but slightly lower than that of At-P4H-2 (Table 2). The Vmax obtained with (Pro-Pro-Gly)10 was 60% of that obtained with poly(L-Pro), Mr 30,000 (Table 2). To study which Pro residues in (Pro-Pro-Gly)10 are preferentially hydroxylated by Cr-P4H-1A, a peptide was partially hydroxylated and subsequently purified from the reaction mixture and subjected to N-terminal sequencing. Surprisingly, the Pro residues in the X positions in the repeating X-Y-Gly triplets were preferentially hydroxylated, while the Y-position Pro residues were hydroxylated to a much lesser degree (Figure 5 ). This hydroxylation preference differs distinctly from that of At-P4H-1, which hydroxylated X-position Pro residues to some extent but acted preferentially on the Y-position Pro residues (Hieta and Myllyharju, 2002Go), and from that of the animal C-P4Hs, which act solely on Y-position Pro residues. The recombinant Cr-P4H-1A also differed from the vertebrate C-P4Hs and the recombinant At-P4H-1 in that it did not show an asymmetrical hydroxylation preference in the peptide but hydroxylated Pro residues quite evenly, excluding the first triplet, which was not hydroxylated at all, and the second and last triplets, in which Pro residues in both positions were hydroxylated very inefficiently (Figure 5).


Figure 5
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  		Figure 5. Analysis of the Hydroxylation of the Pro Residues in (Pro-Pro-Gly)10 by Cr-P4H-1A.

The columns indicate the degree of hydroxylation of the Pro residues in the X- and Y-positions in the X-Y-Gly triplets. P, Pro; G, Gly.

 
Knockdown of the Cr-P4H Gene by RNAi Inhibits Cell Wall Assembly
The effect of suppression of the gene encoding Cr-P4H-1 was studied in C. reinhardtii cells using RNAi. Although the 10 P4H-like genes found in C. reinhardtii do not have an especially high level of DNA sequence identity, the possible existence of homologous 21-bp sequences in Cr-P4H-1 and the nine other P4H-like sequences that could potentially generate identical Dicer products and thus cause unspecific silencing were analyzed. Target recognition in RNAi is a highly sequence-specific process, and nucleotides in the center of the 21-bp stretch are important specificity determinants, with even single nucleotide changes reducing the RNAi effect to undetectable levels (Elbashir et al., 2001Go). Our analysis of the 10 Cr-P4H sequences showed that the C_360012 sequence contains one 21-bp region with three mismatches when compared with the Cr-P4H-1 sequence, with all other 21-bp regions of the nine Cr-P4H sequences having more than three mismatches. Furthermore, the three mismatches in the 21-bp C_360012 sequence were in the center region of the sequence. It is thus evident that expression of only the gene for Cr-P4H-1 was affected in the RNAi experiments.

A hairpin double-stranded RNA construct directed against the Cr-P4H-1 gene transcript was generated in a pSP124S plasmid containing the phleomycin resistance gene ble as a dominant marker for nuclear transformation (Figure 6A ). The plasmid construct was transformed into autolysin-treated wild-type CC125mt+ 137C cells by electroporation, and independent transformant lines were generated from single clones grown on Zeocin plates. In the control experiments, the cells were electroporated with the empty pSP124S plasmid. PCR analysis was used to verify that the Zeocin-resistant RNAi transformants selected for further studies contained the RNAi construct (as shown for one RNAi line in Figure 6B). To study the effect of the Cr-P4H-1 gene knockdown at the protein level, cell extracts were prepared from four independent RNAi lines and from three control lines transformed with the empty vector and analyzed for P4H activity with poly(L-Pro) as a substrate. The P4H activity level of the RNAi strains was found to be reduced to 33 to 58% of that of the controls (Table 3 ).


Figure 6
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  		Figure 6. Construct for Generating a Double-Stranded RNA Hairpin against the Gene Transcript Encoding Cr-P4H-1 and PCR Analysis of the Presence of the RNAi Construct in Transformed Cells.

(A) The sense and antisense strands of part of the coding sequence for Cr-P4H-1 (the antisense long arm is written backward) and the phleomycin resistance gene (ble) are under the control of the C. reinhardtii ribulose bisphosphate carboxylase/oxygenase (RBCS2) promoter.

(B) PCR analysis of the presence of the RNAi construct in one control line transformed with the empty vector and one Cr-P4H-1 RNAi line (RNAi-1). Primer pairs 1 and 2 described in Methods were used to amplify 950- and 750-bp regions of the RNAi hairpin construct, respectively, using genomic DNA isolated from the transformed cells as a template. Amplification using ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) primers was used as a control for analysis of the amounts of template DNA.

 

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  		Table 3. Levels of P4H Activity in Cr-P4H-1 RNAi Lines

 
To study the phenotypic effect of the Cr-P4H-1 knockdown, we analyzed the ultrastructures of the wild type, control, and two independent RNAi lines by transmission electron microscopy of high-pressure frozen and freeze-substituted samples. Silencing of the Cr-P4H-1 expression led to a clear defect in the cell wall (Figure 7 , Table 4 ). The wild-type (data not shown) and control cells were surrounded by an intact, well-defined cell wall, the plasma membrane being closely affixed to the wall (Figure 7A). The walls of the RNAi-treated cells were less clearly defined than those of the control cells, and in some regions, the plasma membrane was not tightly apposed to the wall (Figure 7B). Electron microscopy analyses have shown that the C. reinhardtii cell wall is composed of five structurally distinct layers: the innermost W1 and outermost W7 layers, which are composed of loose networks of fibers, and a prominent central triplet consisting of two dense layers (W2 and W6) and a granular layer (W4) between them (Roberts et al., 1972Go; Goodenough and Heuser, 1985Go; Adair and Snell, 1990Go). All these cell wall layers, with one exception, were clearly visible in the wild-type cells at higher magnifications (Figure 7C). Only the middle layer of the central triplet, W4, which has earlier been shown to consist of granular components rather than fibrous elements (Goodenough and Heuser, 1985Go), was not visible. This may be due to differences between the fixation methods used here and in earlier studies. Silencing of the Cr-P4H-1 expression led to a clear defect in the cell walls, which lacked the typical multilayered structure so that they appeared to consist solely of a relatively homogeneous, loose network of fibrils resembling the W1 and W7 layers of the wild-type cell wall, the layers forming the central triplet being completely absent (Figures 7D to 7F). Quantitative analysis of the cell wall phenotypes showed that 95% of the cells in the control lines transformed with the empty plasmid had a normal wall structure, and detachment of the plasma membrane from the wall was not detected (Table 4). By contrast, 84 to 88% of the cells in two independent RNAi lines had an abnormal wall and/or detachment of the plasma membrane (Table 4).


Figure 7
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  		Figure 7. Transmission Electron Micrographs of High-Pressure Frozen and Freeze-Substituted Samples of C. reinhardtii Cells Electroporated with a Double-Stranded RNA Construct against Cr-P4H-1.

(A) Wild-type control cells transformed with the empty pSP124S plasmid show a clearly defined, multilayered cell wall. Bar = 1000 nm.

(B) The wall is less well defined and uniform in a typical Cr-P4H-1 knockdown cell. Bar = 1000 nm.

(C) The distinct cell wall layers (labeled W1, W2, W6, and W7) can be seen in the control cells at higher magnification. Bar = 1000 nm.

(D) to (F) The walls of the knockdown cells are seen at higher magnification to lack the multilayered structure, and in some cells, the wall is clearly detached from the plasma membrane. Bar = 100 nm.

 

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  		Table 4. Quantitative Analysis of the Cell Wall Phenotypes in Electron Microscopy Specimens of Two Independent Cr-P4H-1 RNAi Lines and a Control Line

 
We also analyzed the growth of the RNAi-treated cells by culturing several independent RNAi and control lines for 10 d and counting the number of cells daily using a hemocytometer. No clear difference was observed between the growth rates of the RNAi and control cells (data not shown). It therefore seems that the cell wall defects of the RNAi-treated cells did not have an adverse effect on their ability to grow. Defects in the cell wall do not necessarily lead to growth problems, for example, some strains of the cw-1 mutant of Chlamydomonas monoica have a faster and some slower doubling times relative to the wild-type cells (Fuentes and VanWinkle-Swift, 2003Go).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
METHODS
 REFERENCES
 
The C. reinhardtii P4H family
The data reported here indicate that the C. reinhardtii genome contains a gene family encoding at least 10 P4H-like polypeptides with sequences similar to those in the animal C-P4Hs. This C. reinhardtii family is considerably larger in size than most animal C-P4H families, as vertebrates are known to have only three C-P4Hs, while C. elegans has four (Myllyharju, 2003Go; Myllyharju and Kivirikko, 2004Go). The only known major exception to the sizes of the animal C-P4H families is the D. melanogaster family of ~20 genes encoding C-P4H {alpha}-subunit–like polypeptides (Abrams and Andrew, 2002Go). Members of the animal C-P4H families typically show distinct differences in their expression in various cells and tissues, one of the three vertebrate isoenzymes being expressed especially in chondrocytes and endothelial cells, for example (Annunen et al., 1998Go), and some of the D. melanogaster C-P4Hs only in the salivary gland or mouth part precursor or in the proventriculus or epidermis (Abrams and Andrew, 2002Go). A surprising feature of the C. reinhardtii P4H family is that a single cell type appears to have a number of P4Hs. As gametes could be considered a second cell type in C. reinhardtii, we analyzed the existing Chlamydomonas EST database (http://www.chlamy.org/) to study whether any of the C. reinhardtii P4H-like genes are specifically or preferentially expressed in the gametes. No evidence to support this was found, the majority of the EST clones representing the Cr-P4Hs being from vegetative C. reinhardtii cDNA libraries. The only exception was the gene C_360012, in which case the only published EST sequence was from a gamete cDNA library. All 10 P4H-like polypeptides are likely to have either a cleavable (seven members) or noncleavable (three members) signal peptide, and they all thus probably reside within the endoplasmic reticulum. It therefore seemed likely that these P4Hs may either show distinct differences in their substrate specificities or a substantial redundancy in their function. Our RNAi data, to be discussed below, indicate that at least one of these P4Hs, Cr-P4H-1, has a function that cannot be fully compensated for by those of the other members. Arabidopsis also has a relatively large P4H family, with at least six genes encoding P4H-like polypeptides, two of which have been cloned and characterized and have been shown to have major differences in their substrate specificities (Hieta and Myllyharju, 2002Go; Tiainen et al., 2005Go), but no data are currently available on possible differences in their expression patterns.

The conservation of the residues that bind the iron atom and the C-5 carboxyl group of 2-oxoglutarate suggests that at least nine of the 10 C. reinhardtii polypeptides are active P4Hs, while in the remaining polypeptide, C_800043, the second iron binding His, is replaced by a Glu. This polypeptide is most likely catalytically inactive, as site-directed mutagenesis of At-P4H-1 has shown that a corresponding replacement leads to complete inactivation of the enzyme (Hieta and Myllyharju, 2002Go).

Characteristics of Cr-P4H-1
Cr-P4H-1, which was cloned and characterized here, shows the highest identity among the C. reinhardtii P4H-like polypeptides to the catalytic C-terminal region of the human C-P4H {alpha}(I) subunit and the At-P4Hs 1 and 2. Cr-P4H-1 was found to have two splicing variants, the three C-terminal residues of the shorter variant being replaced by a 47-residue ShK toxin-like extension in the longer one. Similar extensions have been found in At-P4H-2 and a putative rice (Oryza sativa) P4H (Tiainen et al., 2005Go) and in several cnidarian and nematode proteins, including metalloproteinases (Dauplais et al., 1997Go; Pan et al., 1998Go). This extension is present in nine out of the 10 C. reinhardtii P4H-like polypeptides, although two of them lack some of the six Cys residues that form the disulfide bonds that are important for the structure of the ShK toxin, and none of them is likely to be a potassium channel toxin, as they all lack a critical Lys-Tyr diad (Dauplais et al., 1997Go). The ShK toxin-like extension was not required for Cr-P4H-1 activity, as both splicing variants had essentially identical specific activities.

The recombinant Cr-P4H-1 was found to be an active monomer with a molecular weight that agrees with that previously estimated for a partially purified C. reinhardtii P4H based on gel filtration analysis (Kaska et al., 1987Go). In contrast with the At-P4Hs (Hieta and Myllyharju, 2002Go; Tiainen et al., 2005Go), recombinant Cr-P4H-1 is markedly more soluble and can easily be purified essentially to homogeneity by a single gel filtration, which is highly unusual. P4Hs are vital enzymes in two important processes in animal species: the synthesis of collagens and the response to hypoxia. The human C-P4Hs and HIF-P4Hs are regarded as medically important targets for the development of drugs for the treatment of fibrotic and ischemic diseases, respectively, and the development of specific human P4H inhibitors for this purpose would be greatly facilitated by the availability of three-dimensional structures for the relevant enzymes, which are not currently known. Determination of the structures of these P4Hs may be extremely difficult because the C-P4Hs are {alpha}2ß2 tetramers with a high molecular weight (~240 kD) (Kivirikko and Pihlajaniemi, 1998Go; Myllyharju, 2003Go) and because the expression of active recombinant HIF-P4Hs in high amounts in E. coli has turned out to be difficult (McNeill et al., 2002Go; Hirsilä et al., 2003Go; Choi et al., 2005Go). Recombinant Cr-P4H-1 is therefore currently the best candidate for structural studies of a P4H, and its structure could probably be used as a preliminary model for the catalytic domains of other P4Hs.

Recombinant Cr-P4H-1 resembled the other plant P4Hs studied (for a review, see Kivirikko et al., 1992Go) in that it efficiently hydroxylated poly(L-Pro). Plant P4Hs thus differ markedly from the C-P4Hs and HIF-P4Hs, which require -X-Pro-Gly- and -Leu-X-X-Leu-Ala-Pro- sequences, respectively (Kivirikko and Pihlajaniemi, 1998Go; Bruick and McKnight, 2001Go; Epstein et al., 2001Go; Ivan et al., 2002Go; Myllyharju, 2003Go). Plant P4Hs have generally been believed not to hydroxylate a collagen-like peptide (Pro-Pro-Gly)10 at all or to hydroxylate it only very inefficiently (Kivirikko et al., 1992Go). The only exception so far has been At-P4H-1, which hydroxylated this collagen-like peptide fairly efficiently, the Vmax being similar to that obtained with poly(L-Pro), although the Km was at least 30-fold (Hieta and Myllyharju, 2002Go). Cr-P4H-1 also hydroxylated (Pro-Pro-Gly)10, but the Km was very high, >1500 µM, and the Vmax was only 60% of that obtained with poly(L-Pro). Interestingly, sequencing of the (Pro-Pro-Gly)10 partially hydroxylated by Cr-P4H-1 showed that it acted preferentially on Pro residues in the X-positions of the X-Y-Gly repeats, quite the opposite preference from that noted with animal C-P4Hs, which are entirely specific to the Y-position Pro residues and different from that of At-P4H-1, which showed a high, although not absolute, preference for the Y-position Pro residues (Kivirikko et al., 1992Go; Kivirikko and Pihlajaniemi, 1998Go; Hieta and Myllyharju, 2002Go).

The Pro-rich motifs containing 4Hyp residues in C. reinhardtii HRGPs are strings of contiguous Pro residues, Pro residues alternating with other amino acids, and repeats of the -Pro-Pro-Ser-Pro-X- motif (Ferris et al., 2001Go). We showed here that four synthetic peptides representing the -(Ser-Pro)19- and -Pro-Pro-Ser-Pro-X- motifs of the C. reinhardtii HRGPs GP1, and Sag1, Sad1, and A2 all acted as substrates for Cr-P4H-1 with essentially identical Km values, the highest Vmax being obtained with the one representing the -(Ser-Pro)19- motif of GP1. Pro residues preceding Lys but not those following it have been shown to be hydroxylated in higher plants (Kieliszewski and Lamport, 1994Go), whereas hydroxylation of the position following Lys has been detected adjacent to Lys in the V. carteri cell wall glycoprotein SSG 185 (Ertl et al., 1989Go). Single -Ser-Pro-Lys-Pro-Pro- and -Pro-Pro-Lys-Pro-Ser- motifs are present in domain 2 of the C. reinhardtii GP1 and A2 proteins, respectively, the GP1 domain otherwise mostly consisting of -Ser-Pro-Ala-Pro-Pro- motifs and the A2 domain of -Ser-Pro-Ala-Pro-Pro- and -Ser-Pro-Glu-Pro-Pro- motifs (Ferris et al., 2001Go). As Cr-P4H-1 was capable of hydroxylating the (Ser-Pro-Lys-Pro-Pro)5 peptide, although with a relatively low efficiency, the Lys-containing motifs of GP1 and A2 may become hydroxylated in vivo.

In Vivo Function of Cr-P4H-1
Here, we studied the specific in vivo consequences of the lack of a single C. reinhardtii P4H activity, that of Cr-P4H-1, by RNAi suppression of its gene. Inhibition of total P4H activity by 3,4-dehydro-L-Pro previously has been shown to inhibit cell wall assembly and cell division in tobacco protoplasts, indicating that the 4Hyp residues of their HRGPs have an essential function in the formation of a structurally intact cell wall (Cooper et al., 1994Go). In C. reinhardtii, the HRGPs GP1, GP2, and GP3 are constituents of the W6 layer (i.e., the outermost layer of the dense central triplet of the wall) (Goodenough et al., 1986Go; Adair and Snell, 1990Go). The 4Hyp content of GP1, GP2, and GP3 is 32, 15, and 6%, respectively (Goodenough et al., 1986Go), and the 4Hyp residues are conjugated with short-chain oligosaccharides containing arabinose, galactose, and small amounts of mannose and xylose (Adair and Snell, 1990Go). Analysis of the three-dimensional topologies of GP1, GP2, and GP3 has shown that GP1 has a globular head followed by a short, narrow fibrous neck and an ~90-nm long wider fibrous shaft displaying two characteristic kinks, while GP2 and GP3 have globular heads followed by short, fibrous necks and several globular domains (Goodenough et al., 1986Go; Ferris et al., 2001Go). The fibrous regions of these proteins are formed by their 4Hyp-rich motifs, which adopt a poly(L-Pro) type-II helix conformation (Goodenough et al., 1986Go; Ferris et al., 2001Go). The fibrous portions of GP1, GP2, and GP3 are believed to be important for their self-assembly into distinct cell layers (Goodenough and Heuser, 1985Go; Goodenough et al., 1986Go); thus, the 4Hyp residues are critical for the structure and function of these proteins.

Glycosylation of the 4Hyp residues in the fibrous shafts of HRGPs affects their interactive properties and is necessary for stabilization of the poly(L-Pro) type-II helix conformation of the shaft (Ferris et al., 2001Go). Motifs found in 4Hyp-rich sequences of plant proteins are thought to direct 4Hyp glycosyltransferases to add specific sugar residues to specific 4Hyp residues (Shpak et al., 1999Go, 2001Go; Kieliszewski and Shpak, 2001Go). Two glycosylation codes have been suggested in higher plants, with long, branching sugars being attached to alternating residues via O-galactosyl linkages and short, unbranched sugars to contiguous 4Hyp residues via O-arabinosyl linkages (Shpak et al., 2001Go; Zhao et al., 2002Go; Tan et al., 2003Go), and similar glycosylation codes are likely to exist in C. reinhardtii (Ferris et al., 2001Go).

Cr-P4H-1 activity was found to be essential for the assembly of a proper cell wall, as its knockdown led to highly abnormal cell walls lacking the typical multilayered ultrastructure (Roberts et al., 1972Go; Goodenough and Heuser, 1985Go; Adair and Snell, 1990Go). The first elements to emerge during vegetative wall regeneration are long fibers that are identical in morphology to those seen in layers W1 and W7, while the other wall layers assemble later within this primary matrix (Goodenough and Heuser, 1985Go). The loose fibril network seen in the walls of the RNAi-treated cells resembled morphologically the W1 and W7 fibril networks, suggesting that Cr-P4H-1 was essential for a step subsequent to formation of the primary matrix.

Since our recombinant Cr-P4H-1 was found to efficiently hydroxylate synthetic Pro-rich peptides that represented GP1 sequences, the lack of Cr-P4H-1 activity in the RNAi-treated C. reinhardtii protoplasts probably affected the 4Hyp content of GP1, unless some of the other P4Hs also hydroxylated it. The reduced amounts of 4Hyp residues in GP1 probably also affected its glycosylation and, thus, the interactive properties and stability of its fibrous shaft. The Pro-rich motifs of GP2 consist mainly of strings of two to seven contiguous Pro residues separated by another amino acid. Since Cr-P4H-1 hydroxylated poly(L-Pro) efficiently, it most probably also hydroxylates these GP2 sequences. The amino acid sequence of GP3 is currently unknown, but as in GP1 and GP2, its fibrous neck probably consists of a Pro-rich sequence that is likely to act as a substrate for Cr-P4H-1. Thus, the lack of Cr-P4H-1 activity can be expected to have an effect on the hydroxylation of all three major C. reinhardtii outer cell wall HRGPs.

Interestingly, although the C. reinhardtii genome contains nine other genes that encode P4H-like polypeptides, our RNAi data indicate that they cannot fully compensate for the lack of Cr-P4H-1 activity in proper cell wall assembly and are therefore likely to have different substrate specificities and functions. A similar situation exists in Arabidopsis, where At-P4Hs 1 and 2 have been characterized in detail and have been shown to have distinct substrate specificities (Hieta and Myllyharju, 2002Go; Tiainen et al., 2005Go).

The sexual agglutinins Sag1 and Sad1 of C. reinhardtii consist of a large globular head, a long fibrous shaft in a poly(L-Pro) type-II conformation, and a tail hook (Ferris et al., 2005Go). It has been proposed that the adhesion of gametes is initiated by head–head interactions between agglutinins of opposite mating types, but the heads may also contain lectin motifs that recognize carbohydrates in the shafts and thus contribute to the apposition of the interacting flagellae during mating (Ferris et al., 2005Go). Furthermore, distinct amino acid sequence differences between the Pro-rich shafts of Sag1 and Sad1, and hence between their 4Hyp patterns, may lead to the acquisition of distinctive glycosylation patterns (Shpak et al., 1999Go, 2001Go; Zhao et al., 2002Go; Tan et al., 2003Go) that can influence shaft–shaft interactions and, thus, adhesion. Here, we showed that Cr-P4H-1 hydroxylates in vitro the Pro-rich motifs present in the Sag1 and Sad1 agglutinins. It will therefore be of interest to analyze in further studies whether the function of Cr-P4H-1 is essential for mating in C. reinhardtii.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
REFERENCES
 
Strains
The Chlamydomonas reinhardtii wild-type strain CC125 mt+ 137c was obtained from Elisabeth Harris (Chlamydomonas Genetics Center, Duke University, Durham, NC) and was cultured on Tris-acetate-phosphate (TAP) medium by a standard method (Harris, 1989Go) or on 1.5% agar plates. The wild-type strains CC620 mt+ 137c and CC621 mt– 137c (Chlamydomonas Genetics Center) were used for the isolation of gamete autolysin to remove cell walls for C. reinhardtii transformation by electroporation (Harris, 1989Go).

Identification of C. reinhardtii Genes Encoding P4H-Like Polypeptides
A sequence homology search of the C. reinhardtii EST Database indicated the presence of EST sequences (e.g., GenBank accession numbers AW661167 and BG860906) that were similar to the catalytic C-terminal regions of the human C-P4H {alpha}(I) subunit sequence. The overlapping EST sequences could be fused into an open reading frame encoding a 253–amino acid polypeptide, named here Cr-P4H-1, the cleavage site for a signal peptide being predicted using the SignalP server (Nielsen et al., 1997Go). The 5' end of the open reading frame was verified by 5' rapid amplification of cDNA ends PCR of cDNA synthesized from mRNA isolated from cultured wild-type CC125 C. reinhardtii cells. Total RNA was isolated using TRIzol reagent (Gibco BRL) and poly(A)+ mRNA using the Poly(A) Quik mRNA isolation kit (Stratagene), and cDNA was generated using the Smart PCR cDNA synthesis kit (BD Biosciences). Nine other P4H-like sequences were found among the gene annotations in the C. reinhardtii genome draft release, version 2.0, namely, C_360012 (protein ID 163913; 187378 in version 3.0 of the Chlamydomonas genome sequence), C_390100 (protein ID 164489; 166317 in version 3.0), C_150026 (protein ID 156619; 114525 in version 3.0), C_280045 (protein ID 162007; 79261 in version 3.0), C_1000010 (protein ID 152342; 122372 in version 3.0), C_2500005 (protein ID 160996; 138462 in version 3.0), C_50128 (protein ID 167613; 111555 in version 3.0), C_130209 (protein ID 155504; 111255 in version 3.0), and C_800043 (protein ID 170197; 143143 in version 3.0), and in annotation C_110025 (protein ID 153851; 138508 in version 3.0), encoding an alternatively spliced form of Cr-P4H-1. Amino acid sequence alignments for phylogenetic analysis were generated using the ClustalW server (Chenna et al., 2003Go) and were further adjusted manually (see Supplemental Figure 1 online). A neighbor-joining tree was constructed from the aligned sequences using the MEGA program, version 2.1 (Kumar et al., 2001Go), using p-distance and complete deletion for the options. The statistical significance of the resulting tree was tested by means of 1000 bootstrap replications (Felsenstein, 1985Go).

Cloning and Recombinant Expression of C. reinhardtii P4H-1A in Insect Cells
The PCR primers 5'-GCGAGATCTATGCTGCTTTTAGGACTCGTTCTG-3' (BglII site underlined) and 5'-CGCTCTAGATCAATGACGCCCTCCGATGG-3' (XbaI site underlined) were synthesized based on the cDNA sequence encoding Cr-P4H-1A and used to obtain a 762-bp PCR product from a wild-type C. reinhardtii cDNA library (Chlamydomonas Genetics Center). The PCR template was prepared by incubating 2 µL of the cDNA library in a 200-µL final volume in 1% Nonidet P-40, 100 µg/mL proteinase K, 1 mM EDTA, and 10 mM Tris, pH 8.0, at 55°C for 1 h, followed by 10 min at 95°C and centrifuged at 12,000 rpm for 5 min. Ten microliters of the prepared template was used in a 50-µL PCR reaction that involved 30 cycles of denaturation for 1 min at 94°C, annealing for 2 min at 55°C, and extension for 3 min at 72°C, followed by a final incubation at 72°C for 10 min. To increase the amount of PCR product, a second PCR reaction was performed with 20 µL of the first product diluted 1:50 as the template, the cycles being as above. The resulting fragment was digested with BglII and XbaI and cloned into a similarly digested pVL1392 baculovirus vector (Pharmingen). The sequence was verified on an automated DNA sequencer (ABI Prism 377; Applied Biosystems).

The recombinant vector was cotransfected into Spodoptera frugiperda Sf9 cells with BaculoGold DNA (Pharmingen) by calcium phosphate transfection, and the recombinant viruses were amplified (Crossen and Gruenwald, 1998Go). Sf9 insect cells (Invitrogen) were cultured as monolayers in TNM-FH medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (BioClear) or suspended in Sf900IISFM serum-free medium (Invitrogen). The cells were seeded at a density of 5 x 106/100-mm plate or 106/mL and infected at a multiplicity of 5 with the virus coding for the Cr-P4H-1 polypeptide. The cells were harvested 72 h after infection and washed with a solution of 0.15 M NaCl and 0.02 M phosphate, pH 7.4, homogenized in 0.1 M NaCl, 0.1 M glycine, 10 µM DTT, 0.1% Triton X-100, and 0.01 M Tris buffer, pH 7.8, and centrifuged at 10,000g for 20 min. The pellets were further solubilized in 1% SDS, and aliquots of all soluble fractions were analyzed by 12% SDS-PAGE under reducing conditions.

Expression of Recombinant C. reinhardtii P4H-1A and P4H-1B in Escherichia coli
The PCR primers 5'-GGAATTCCATATGGCGCCTTCAAGCGCCATGAT-3' (NdeI site underlined) and 5'-CGCAGATCTTCAATGACGCCCTCCGATGG-3' or 5'-CGCAGATCTTTAGGTGCACTTCTTGCACGAG (BglII sites underlined) were used to amplify the cDNAs encoding Cr-P4H-1A and Cr-P4H-1B without the signal sequences and with flanking NdeI and BglII sites, and the products were cloned into a NdeI-BamHI–digested bacterial expression vector pET15b in frame with an N-terminal His tag (Novagen). The expression plasmids were transformed into the E. coli Origami (DE3) strain (Novagen). The cells were grown at 37°C to an optical density of 0.8 at 600 nm and incubated at 20°C for 30 min, and expression was induced with 0.5 mM isopropyl-1-thio-ß-D-galactopyranoside. The cells were harvested after overnight induction at 20°C, suspended in 0.1 volume of 0.1 M NaCl, 0.1 M glycine, 10 µM DTT, and 10 mM Tris buffer, pH 7.8, disrupted by sonication, and centrifuged at 17,000g for 20 min, and the soluble and insoluble fractions were analyzed by 12% SDS-PAGE.

Purification of the Recombinant C. reinhardtii P4H-1 Polypeptides
The recombinant Cr-P4H-1A polypeptide expressed in insect cell suspension cultures was purified by applying the soluble fraction of the cell homogenate to a HiPrep Sephacryl S-100 HR gel filtration column (GE Healthcare). The eluted fractions were analyzed by 12% SDS-PAGE, and those containing the pure recombinant protein were pooled. Soluble proteins from the E. coli expressions were applied to a 30-mL Ni-NTA chelating Sepharose column (GE Healthcare) equilibrated with 0.1 M NaCl, 0.1 M glycine, 10 µM DTT, and 10 mM Tris buffer, pH 7.8. The bound proteins were eluted with a 200-mL linear imidazole gradient (0 to 0.5 M), and the fractions were analyzed by 12% SDS-PAGE. The fractions containing the recombinant Cr-P4H-1A or Cr-P4H-1B polypeptides were pooled and concentrated using Amicon Ultra-15 10K concentrators (Millipore), after which a 1-mL sample was loaded onto a SuperDex 75 HR (GE Healthcare) column equilibrated with the above buffer. Fractions of 2 mL were collected and analyzed by 12% SDS-PAGE, and those containing the pure Cr-P4H-1A or Cr-P4H-1B were pooled.

CD Spectroscopy
CD spectra were recorded with a JASCO J-715 CD spectropolarimeter, where the sample cell temperature was controlled by a JASCO PFD-350S Peltier-type temperature control unit. Thermal denaturation of the purified recombinant Cr-P4H-1A was followed by continuous monitoring of ellipticity changes at a fixed wavelength of 206 nm while the sample was heated at a constant rate of 30°C/h. The protein concentration in the sample was 0.2 mg/mL in 10 mM sodium phosphate buffer, pH 6.8.

RNAi
The pSP124S plasmid was used to generate a hairpin Cr-P4H-1-RNAi construct, as shown in Figure 6A. The RBCS2 promoter region was amplified by PCR from the pSP124S with the primers 5'-ATAAGAATGCGGCCGCGATTTAAATGCCAGAAGGAGC-3' (NotI site underlined) and 5'-CGCGGATCCTTTAAGATGTTGAGTGACTTCTC-3' (BamHI site underlined). The PCR was performed in 30 cycles of 94°C for 1 min, 58°C for 2 min, and 72°C for 3 min, and the product of the RBCS2 promoter was cloned into NotI-BamHI–digested pSP124S to generate a vector with an additional copy of the promoter. The sense and antisense strands of the coding sequence for Cr-P4H-1 were amplified by PCR using the primers 5'-GCGGGATCCATGCTGCTTTTAGGACTCGTTCTGGCC-3' (BamHI site underlined), 5'-CAGGTACATGAGCATCGTGACCAC-3' and 5'-CGGAATTCATGCTGCTTTTAGGACTCGTTCTGGCC-3' (EcoRI sites underlined), and 5'-ACTTGTCACCCTTAAGCGTG-3' using the pVL1392-Cr-P4H-1A vector generated above as a template. The products obtained were digested and cloned into BamHI-EcoRI–digested pSP124S with two copies of the RBCS2 promoter. The cloned sequences were confirmed by DNA sequencing as above. The hairpin Cr-P4H-1-RNAi construct was transformed into autolysin-treated C. reinhardtii wild-type strain CC125+ cells by electroporation at an electrical strength of 1375 V/cm in a transformation medium of 40 mM sucrose in water (Shimogawara et al., 1998Go). The bacterial phleomycin resistance gene ble was used for selection on Zeocin plates. The cells were incubated on a TAP agar plate with Zeocin (10 µg/mL) for 7 d at room temperature. Transformant cells electroporated with the pSP124S plasmid without the hairpin RNAi construct were used as a control as well as wild-type cells electroporated without any plasmid DNA. Single colonies of transformants were picked up and grown in TAP medium with Zeocin (10 µg/mL) for further analysis.

Analysis of the Presence of the RNAi Construct in the Transformed Lines
Total genomic DNA was isolated from the transformed lines as described previously (http://www.biology.duke.edu/chlamy/methods/quick_pcr.html). A DNA pellet of 5 to 10 µL in size was resuspended in 50 µL of 10 mM Na-EDTA by vortexing, incubated at 100°C for 5 min, vortexed, and centrifuged at 12,000g for 1 min. A 3-µL fraction of the supernatant was used as a template in the PCR reactions. The primers 5'-TTTAAGATGTTGAGTGACTTCTC-3' and 5'-ACTTGTCACCCTTAAGCGTG-3' (primer pair 1 in Figure 6B) were used to amplify a 750-bp fragment extending from the upstream RBCS2 promoter to the 3' end of the sense strand of Cr-P4H-1, and primers 5'-GATTTAAATGCCAGAAGGAGC-3' and 5'-CAGGTACATGAGCATCGTGACCA-3' (primer pair 2 in Figure 6B) were used to amplify a 950-bp fragment extending from the downstream RBCS2 promoter to the 3' end of the antisense strand of Cr-P4H-1. A PCR fragment amplified from the coding region of the Rubisco gene was used to analyze the amount of template between the samples.

Transmission Electron Microscopy of High-Pressure Frozen and Freeze-Substituted C. reinhardtii Cell Specimens
Samples of wild-type, control, and RNAi-treated C. reinhardtii cells were spread on a Millipore filter (size 45 µm), and the moist cell paste was transferred to specimen carriers (Leica) and cryofixed in a Leica EM Pact high-pressure freezer. Specimens were also freeze-substituted (Walther and Ziegler, 2002Go) in a Leica AFS freeze substitution system for Epon embedding. The specimens were infiltrated in 1.7% osmium tetroxide and 0.1% uranyl acetate in acetone containing 5% water for 10 h at –90°C and gradually warmed to 0°C. They were then removed from the specimen carriers and infiltrated into Epon Ember 812 (Electron Microscopy Sciences) at room temperature and polymerized at 60°C for 48 h. Thin sections were cut with a Leica Ultracut UCT ultramicrotome, followed by staining in uranyl acetate and lead citrate, and examined in a Philips CM100 transmission electron microscope. Images were captured with a CCD camera equipped with TCL-EM-Menu, version 3 (Tietz Video and Image Processing Systems). Phenotypic characterization of two independent Cr-P4H-1 RNAi and control lines was performed by analyzing 71 mutant and 42 control cells arbitrarily chosen in the transmission electron microscopy specimens.

Other Assays
The molecular weight of the recombinant Cr-P4H-1A polypeptide was analyzed by gel filtration on a calibrated HiPrep Sephacryl S-100 HR column (GE Healthcare). P4H activity was assayed with a modification of a standard method based on the hydroxylation-coupled decarboxylation of 2-oxo-[1-14C]glutarate (Kivirikko and Myllylä, 1982Go) at 30°C. The Tris-HCl buffer was replaced with 50 mM HEPES, pH 6.8, and the concentration of FeSO4 was increased to 200 µM (Kaska et al., 1987Go). Poly(L-Pro) was from Sigma-Aldrich and (Pro-Pro-Gly)10 from the Peptide Institute, while all the other synthetic peptides were from Innovagen. All the peptides, except poly(L-Pro), were denatured by heating to 100°C for 10 min, followed by rapid cooling before addition to the enzyme reaction mixture. Km values were determined as described previously (Myllyharju and Kivirikko, 1997Go). The partially hydroxylated (Pro-Pro-Gly)10 peptide was purified from the reaction mixture and sequenced as described previously (Hieta and Myllyharju, 2002Go).

Preparation of cell extracts from RNAi and control lines for P4H activity assay was performed as described previously (Kaska et al., 1987Go). Briefly, 25 x 106 cells were harvested by centrifugation (1000g) and were treated with autolysin for 1 h at room temperature. The resulting protoplasts were washed twice by resuspension in 1x TAP medium and centrifuged at 1000g, and the final cell pellet was resuspended in 2 volumes of ice-cold solubilization buffer (50 mM Tris-HCl, pH 7.8, at 4°C, 300 mM KCl, 20 mM MgCl2, 0.05 mM DTT, and 0.1% Triton X-100). After incubation for 1 h at 4°C, the solution was centrifuged at 15,000g for 20 min, and the supernatants were analyzed for P4H activity with poly(L-Pro) as a substrate as above.

The growth of several RNAi and control lines was studied by suspending an equal number of cells in 1 mL of TAP medium containing 10 µg/mL Zeocin and culturing using standard methods for 10 d. The cells were counted daily with a hemocytometer.

Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: Cr-P4H-1A (AW661167 and BG860906), GP1 (AAG45420), GP2 (AY596305), Sag1 (AY450930), Sad1 (AY450929), and A2 (AAG45421). Sequence data also can be found in the C. reinhardtii genome draft release, versions 2.0 and 3.0, under the following accession number: Cr-P4H-1B (C_110025, protein ID 153851 in 2.0, 138508 in 3.0). The accession numbers for the nine other C. reinhardtii P4H-like polypeptides are given in Figure 1B.

Supplemental Data
The following material is available in the online version of this article.

Supplemental Figure 1. Sequence Alignments Used in Phylogenetic Analysis.


   Acknowledgments
 
We thank Merja Nissilä and Liisa Äijälä (University of Oulu) for their expert technical assistance, Elizabeth Harris (Duke University, Durham, NC) for providing the C. reinhardtii strains and the cDNA library, and Saul Purton (University College, London, UK) for providing the pSP124S plasmid. Research was supported by the Health Science Council (Grants 200471 and 202469), by the Finnish Centre of Excellence Program 2000 to 2005 of the Academy of Finland (Grant 44843), and by the S. Juselius Foundation.


   Footnotes
 
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Johanna Myllyharju (johanna.myllyharju{at}oulu.fi).

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www.plantcell.org/cgi/doi/10.1105/tpc.106.042739

Received March 22, 2006; Revision received November 14, 2006. accepted December 5, 2006.


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TOP
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 INTRODUCTION
 RESULTS
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