Chlamydomonas reinhardtii Has Multiple Prolyl4-Hydroxylases, One of Which Is Essential for ProperCell Wall Assembly W

Katriina Keskiaho,a Reija Hieta,a Raija Sormunen,b and Johanna Myllyharjua,1

a Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu,

FIN-90014 Oulu, Finlandb Biocenter Oulu and Department of Pathology, University of Oulu, FIN-90014 Oulu, Finland

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

4-Hydroxyproline (4Hyp) is found in many Hyp-rich plant glyco-

proteins (HRGPs) that are the major structural components of cell

walls (for reviews, see Cassab, 1998; Kieliszewski and Shpak,

2001). The HRGP family contains three major subgroups: repet-

itive Pro-rich proteins, extensins, and arabinogalactan proteins

(Cassab, 1998; Kieliszewski and Shpak, 2001). The cell walls in

green algae are almost entirely built up from extensin-like HRGPs,

the Chlamydomonas reinhardtii cell wall containing 25 to 30 dif-

ferent HRGPs (Adair and Snell, 1990; Sumper and Hallmann,

1998). In addition, sexual adhesion of C. reinhardtii gametes

during mating occurs via agglutinins that are also HRGPs (Ferris

et al., 2005). Three classes of Pro-rich motifs that consist of con-

tiguous 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 subsequently4-hydroxylated (Adair and Snell, 1990; Sumper

and Hallmann, 1998; Ferris et al., 2001).

Most of the 4Hyp in animal proteins is found in collagens and

>20 additional proteins with collagen-like sequences. 4Hyp res-

idues play a critical role in all collagens, as they are essential for

the formation of stable collagen triple helices at body tempera-

ture (for reviews, see Kivirikko and Pihlajaniemi, 1998; Myllyharju,

2003; Myllyharju and Kivirikko, 2004).

The formation of 4Hyp in all these proteins is catalyzed by

prolyl 4-hydroxylases (P4Hs), which act on Pro residues in pep-

tide linkages and require Fe2þ, 2-oxoglutarate, O2, and ascorbate

(Kivirikko and Pihlajaniemi, 1998; Myllyharju, 2003). The well-

characterized vertebrate type I, II, and III collagen P4Hs (C-P4Hs)

reside within the lumen of the endoplasmic reticulum and are

a2b2 tetramers in which the catalytic a subunits have three iso-

forms: a(I), a(II), and a(III) (Helaakoski et al., 1989; Annunen et al.,

1997; Kukkola et al., 2003; Van Den Diepstraten et al., 2003).

Animal C-P4Hs have also been identified and characterized from

nematodes (Veijola et al., 1994; Friedman et al., 2000; Winter

and Page, 2000; Merriweather et al., 2001; Myllyharju et al.,

2002; Riihimaa et al., 2002; Winter et al., 2003) and Drosophila

melanogaster (Annunen et al., 1999; Abrams and Andrew, 2002).

A second family of animal P4Hs has a cytoplasmic and nuclear

location. It consists of three isoenzymes with no b subunit that

play a central role in the oxygen-dependent regulation of the

hypoxia-inducible transcription factor HIF (Bruick and McKnight,

2001; Epstein et al., 2001; Ivan et al., 2002). These HIF-P4Hs are

effective oxygen sensors due to their high Km values for O2

(Hirsila et al., 2003).

Plant P4Hs have been partially purified and characterized from

many higher plant sources (for a review, see Kivirikko et al., 1992)

and the green algae C. reinhardtii and Volvox carteri (Kaska et al.,

1987, 1988), but the only plant P4Hs cloned and characterized so

far are two monomeric enzymes, At-P4Hs 1 and 2, from Arabi-

dopsis thaliana and one from Nicotiana tabacum (Hieta and

1 To whom correspondence should be addressed. E-mail johanna.myllyharju@oulu.fi; fax 358-8-537 5811.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Johanna Myllyharju(johanna.myllyharju@oulu.fi).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.106.042739

The Plant Cell, Vol. 19: 256–269, January 2007, www.plantcell.org ª 2007 American Society of Plant Biologists

Myllyharju, 2002; Tiainen et al., 2005; Yuasa et al., 2005). 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 differ-

ences in their substrate specificities (Hieta and Myllyharju, 2002;

Tiainen et al., 2005). 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., 1999).

The overall amino acid sequence identities between the

a subunits of animal C-P4Hs and the HIF-P4Hs and plant P4Hs

are relatively low, but the catalytically critical residues are con-

served (Myllyharju, 2003). 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 represent-

ing 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

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., 1987, 1988), 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 a(I) subunit (Helaakoski et al., 1989)

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 a(I) subunit, the identity

with the two low molecular weight P4Hs from Arabidopsis (Hieta

and Myllyharju, 2002; Tiainen et al., 2005) being 40% and that

with the PBCV-1 P4H (Eriksson et al., 1999) 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 Fe2þ atom and the Lys that

binds the C-5 carboxyl group of the 2-oxoglutarate at the cat-

alytic site in the C-P4Hs, At-P4Hs, and PBCV-1 P4H (Lamberg

et al., 1995; Myllyharju and Kivirikko, 1997; Eriksson et al., 1999;

Hieta and Myllyharju, 2002; Tiainen et al., 2005) 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-oxoglu-

tarate to the Fe2þ atom and the decarboxylation of this cosub-

strate (Myllyharju and Kivirikko, 1997), 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., 1999; Hieta and Myllyharju,

2002). The Cr-P4H-1 polypeptide contains three Cys residues,

which are all conserved in At-P4H-1 (Figure 1A).

No other P4H-like C. reinhardtii sequences were found in

GenBank. However, analysis of the annotations in the C. rein-

hardtii 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 polypep-

tides (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 exten-

sion is homologous to ShK, a 35-residue polypeptide potassium

channel toxin from the sea anemone Stichodactyla helianthus

(Dauplais et al., 1997; Pan et al., 1998), a similar homologous

region having also been found previously in At-P4H-2 (Tiainen

et al., 2005). 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 a-subunits of the vertebrate and C. elegans C-P4Hs, the

algal and plant P4Hs are more closely related to the human,

mouse, and rat a(III) subunit isoforms than to the a(I) and a(II)

subunits or to the C. elegans a-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.

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

Chlamydomonas Prolyl 4-Hydroxylases 257

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

258 The Plant Cell

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 sin-

gle-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.

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 208C 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 808C, the melting temperature being ;588C

(Figure 4). These results indicate that the recombinant polypep-

tide has a fairly compact, stable fold.

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 mM 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., 1992). As

the specific activities of Cr-P4H-1A and Cr-P4H-1B were iden-

tical, 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 Fe2þ was 30 mM, 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 mM, 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 mM, was 15-fold lower than the

corresponding values for the At-P4Hs 1 and 2 and the PBCV-1

P4H (Table 1).

The 2-oxoglutarate analogs pyridine-2,4-dicarboxylate and

pyridine-2,5-dicarboxylate, which are effective competitive in-

hibitors of vertebrate C-P4Hs, were found to inhibit Cr-P4H-1,

but only at relatively high concentrations, the Ki values being 70

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 a subunits from human [Hs a(I), a(II), and a(III)],

mouse [Mm a(I), a(II), and a(III)], rat [Rn a(I) and a(III)], chicken [Gg a(I)], C.

elegans (Ce PHY-1, PHY-2, and PHY-3), Brugia malayi (Bm PHY-1),

Onchocerca volvulus (Ov PHY-1), D. melanogaster [Dm a(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. 2001). Bootstrap values determined from 1000 replications are

shown at each node. Bar ¼ 10% divergence.

Figure 1. (continued).

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 a(I) subunit [Hs a(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.

Chlamydomonas Prolyl 4-Hydroxylases 259

and 100 mM, 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. Zn2þ was

found to be quite an effective inhibitor with respect to Fe2þ, the Ki

value being 10 mM, which is nevertheless markedly higher than

the Ki of 0.6 mM determined for human C-P4H-I (Kivirikko et al.,

1992).

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 mM, 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 Myllyla, 1982; Kivirikko and

Pihlajaniemi, 1998; Myllyharju, 2003). 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., 1987).

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., 2001). The motifs

may be strings of contiguous Pro residues or Pro residues al-

ternating 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., 2001, 2005)

all served as substrates for Cr-P4H-1A with essentially identical

Km values, ;100 mM, 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 mM, 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,

1994). 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 mM (Eriksson

et al., 1999).

The Cr-P4H-1A enzyme also hydroxylated a denatured colla-

gen-like (Pro-Pro-Gly)10 peptide, but very inefficiently, with a Km

of >1500 mM, 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 reac-

tion mixture and subjected to N-terminal sequencing. Surpris-

ingly, 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 ex-

tent but acted preferentially on the Y-position Pro residues (Hieta

Figure 3. Expression of Recombinant Cr-P4H-1A and Cr-P4H-1B Poly-

peptides 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]).

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 908C. mrw, mean residue

weight.

260 The Plant Cell

and Myllyharju, 2002), 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).

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 ho-

mologous 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.,

2001). Our analysis of the 10 Cr-P4H sequences showed that the

C_360012 sequence contains one 21-bp region with three mis-

matches 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 con-

struct was transformed into autolysin-treated wild-type CC125mtþ137C 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 knock-

down 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).

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 mem-

brane was not tightly apposed to the wall (Figure 7B). Electron

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

Km or Ki (mM)

Cosubstrate or Inhibitor Constant Cr-P4H-1 At-P4H-1a At-P4H-2b PBCV-1 P4Hc

Fe2þ Km 30 16 5 0.4

2-Oxoglutarate Km 250 130 170 20.0

Ascorbate Km 20 300 300 300.0

Poly(L-Pro)

Mr ¼ 5,000 to 10,000 Km 140 2 30 NDd

Mr ¼ 30,000 to 40,000 Km 25 ND 13 100.0

Pyridine-2,4-dicarboxylate Ki 70 ND 10 ND

Pyridine-2,5-dicarboxylate Ki 100 ND 80 ND

Zn2þ Ki 10 ND ND ND

a Hieta and Myllyharju (2002).b Tiainen et al. (2005).c Eriksson et al. (1999).d ND, not determined.

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

Pro-Rich Peptides

Substrate Km (mM) Vmax (%)

Poly(L-Pro), Mr 30,000 25 100

Poly(L-Pro), Mr 5,000 140 90

(Ser-Pro)19a 120 90

(Ser-Pro-Ala-Pro-Pro)5b 100 65

(Ser-Pro-Glu-Pro-Pro)5b 100 40

(Ser-Pro-Lys-Pro-Pro)5b 100 25

(Pro-Pro-Gly)10 >1,500 60

a 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 sequence present in the C. reinhardtii

GP1 protein.b Motifs present in the C. reinhardtii GP1, Sag1, Sad1, and A2 proteins.

Chlamydomonas Prolyl 4-Hydroxylases 261

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 net-

works 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., 1972; Goodenough and Heuser, 1985; Adair

and Snell, 1990). 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, 1985), was not

visible. This may be due to differences between the fixation meth-

ods 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 re-

sembling the W1 and W7 layers of the wild-type cell wall, the

layers forming the central triplet being completely absent (Fig-

ures 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).

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

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.

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.

262 The Plant Cell

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, 2003).

DISCUSSION

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 polypep-

tides with sequences similar to those in the animal C-P4Hs. This

C. reinhardtii family is considerably larger in size than most ani-

mal C-P4H families, as vertebrates are known to have only three

C-P4Hs, while C. elegans has four (Myllyharju, 2003; Myllyharju

and Kivirikko, 2004). 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 a-subunit–like polypeptides (Abrams

and Andrew, 2002). 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., 1998), 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, 2002). 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 evi-

dence 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 mem-

bers) 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

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

RNAi Line Activity (%)a

RNAi-1 33 6 15

RNAi-2 46 6 10

RNAi-3 58 6 15

RNAi-4 51 6 24

a The P4H activity level is given as a percentage relative to that of at

least three independent control lines. The values are means 6 SD from

at least three independent experiments.

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 ¼ 1000 nm.

(B) The wall is less well defined and uniform in a typical Cr-P4H-1 knockdown cell. Bar ¼ 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 ¼ 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 ¼ 100 nm.

Chlamydomonas Prolyl 4-Hydroxylases 263

these P4Hs, Cr-P4H-1, has a function that cannot be fully com-

pensated 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, 2002; Tiainen

et al., 2005), but no data are currently available on possible dif-

ferences 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 cata-

lytically 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, 2002).

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 a(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., 2005) and

in several cnidarian and nematode proteins, including metal-

loproteinases (Dauplais et al., 1997; Pan et al., 1998). 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., 1997). 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 mono-

mer 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., 1987). In contrast with the At-

P4Hs (Hieta and Myllyharju, 2002; Tiainen et al., 2005), recom-

binant 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 inhib-

itors for this purpose would be greatly facilitated by the avail-

ability 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

a2b2 tetramers with a high molecular weight (;240 kD) (Kivirikko

and Pihlajaniemi, 1998; Myllyharju, 2003) and because the ex-

pression of active recombinant HIF-P4Hs in high amounts in

E. coli has turned out to be difficult (McNeill et al., 2002; Hirsila

et al., 2003; Choi et al., 2005). Recombinant Cr-P4H-1 is there-

fore 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 stud-

ied (for a review, see Kivirikko et al., 1992) 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 Pihla-

janiemi, 1998; Bruick and McKnight, 2001; Epstein et al., 2001;

Ivan et al., 2002; Myllyharju, 2003). 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., 1992). The only exception so far has been At-

P4H-1, which hydroxylated this collagen-like peptide fairly effi-

ciently, the Vmax being similar to that obtained with poly(L-Pro),

although the Km was at least 30-fold (Hieta and Myllyharju, 2002).

Cr-P4H-1 also hydroxylated (Pro-Pro-Gly)10, but the Km was very

high, >1500 mM, and the Vmax was only 60% of that obtained with

poly(L-Pro). Interestingly, sequencing of the (Pro-Pro-Gly)10 par-

tially hydroxylated by Cr-P4H-1 showed that it acted preferen-

tially 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 resi-

dues 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., 1992; Kivirikko and Pihlajaniemi, 1998; Hieta and

Myllyharju, 2002).

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., 2001). 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 essen-

tially 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, 1994),

whereas hydroxylation of the position following Lys has been

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

Number of

Cells Analyzeda

Normal Cell

Wall (%)

Cell Wall Abnormality

Cell Line I (%)b II (%)c III (%)d

Control 42 95 – 5 –

RNAi-1 34 12 3 82 3

RNAi-2 37 16 24 27 33

a Cells were arbitrarily picked from the electron microscopy specimens,

and their walls were analyzed visually.b Apparently normal cell wall detached from the plasma membrane.c Non-multilayered cell wall.d Non-multilayered cell wall detached from the plasma membrane.

264 The Plant Cell

detected adjacent to Lys in the V. carteri cell wall glycoprotein

SSG 185 (Ertl et al., 1989). 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., 2001). 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., 1994).

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., 1986; Adair and Snell,

1990). The 4Hyp content of GP1, GP2, and GP3 is 32, 15, and

6%, respectively (Goodenough et al., 1986), and the 4Hyp res-

idues are conjugated with short-chain oligosaccharides contain-

ing arabinose, galactose, and small amounts of mannose and

xylose (Adair and Snell, 1990). 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.,

1986; Ferris et al., 2001). 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., 1986; Ferris et al.,

2001). The fibrous portions of GP1, GP2, and GP3 are believed

to be important for their self-assembly into distinct cell layers

(Goodenough and Heuser, 1985; Goodenough et al., 1986); 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., 2001). 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., 1999, 2001; Kieliszewski and Shpak, 2001). Two glycosy-

lation 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 contig-

uous 4Hyp residues via O-arabinosyl linkages (Shpak et al.,

2001; Zhao et al., 2002; Tan et al., 2003), and similar glycosy-

lation codes are likely to exist in C. reinhardtii (Ferris et al., 2001).

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., 1972; Goodenough and Heuser, 1985; Adair and Snell,

1990). 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, 1985). 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 af-

fected 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 sep-

arated 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 ex-

pected 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, 2002;

Tiainen et al., 2005).

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., 2005). 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 carbo-

hydrates in the shafts and thus contribute to the apposition of the

interacting flagellae during mating (Ferris et al., 2005). Further-

more, 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., 1999, 2001; Zhao et al., 2002; Tan et al.,

2003) 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

Strains

The Chlamydomonas reinhardtii wild-type strain CC125 mtþ 137c was

obtained from Elisabeth Harris (Chlamydomonas Genetics Center, Duke

Chlamydomonas Prolyl 4-Hydroxylases 265

University, Durham, NC) and was cultured on Tris-acetate-phosphate

(TAP) medium by a standard method (Harris, 1989) or on 1.5% agar

plates. The wild-type strains CC620 mtþ 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 elec-

troporation (Harris, 1989).

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 a(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., 1997). The 59 end of the open reading frame was verified

by 59 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)þ 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., 2003) 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., 2001), 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, 1985).

Cloning and Recombinant Expression of C. reinhardtii P4H-1A

in Insect Cells

The PCR primers 59-GCGAGATCTATGCTGCTTTTAGGACTCGTTCTG-39

(BglII site underlined) and 59-CGCTCTAGATCAATGACGCCCTCCGA-

TGG-39 (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 mL of

the cDNA library in a 200-mL final volume in 1% Nonidet P-40, 100 mg/mL

proteinase K, 1 mM EDTA, and 10 mM Tris, pH 8.0, at 558C for 1 h,

followed by 10 min at 958C and centrifuged at 12,000 rpm for 5 min. Ten

microliters of the prepared template was used in a 50-mL PCR reaction

that involved 30 cycles of denaturation for 1 min at 948C, annealing for

2 min at 558C, and extension for 3 min at 728C, followed by a final

incubation at 728C for 10 min. To increase the amount of PCR product, a

second PCR reaction was performed with 20 mL 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, 1998). Sf9 insect cells (Invitrogen) were cultured as mono-

layers 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 3 106/100-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 mM 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 59-GGAATTCCATATGGCGCCTTCAAGCGCCATGAT-39

(NdeI site underlined) and 59-CGCAGATCTTCAATGACGCCCTCCGA-

TGG-39 or 59-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 bac-

terial 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 378C to an optical

density of 0.8 at 600 nm and incubated at 208C for 30 min, and expression

was induced with 0.5 mM isopropyl-1-thio-b-D-galactopyranoside. The

cells were harvested after overnight induction at 208C, suspended in 0.1

volume of 0.1 M NaCl, 0.1 M glycine, 10 mM 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 sus-

pension 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 mM 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 ellip-

ticity changes at a fixed wavelength of 206 nm while the sample was

heated at a constant rate of 308C/h. The protein concentration in the

sample was 0.2 mg/mL in 10 mM sodium phosphate buffer, pH 6.8.

266 The Plant Cell

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 59-ATAAG-

AATGCGGCCGCGATTTAAATGCCAGAAGGAGC-39 (NotI site under-

lined) and 59-CGCGGATCCTTTAAGATGTTGAGTGACTTCTC-39 (BamHI

site underlined). The PCR was performed in 30 cycles of 948C for 1 min,

588C for 2 min, and 728C 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 59-GCGGGATCCATGCTGCTTTTAGGACTCGTTC-

TGGCC-39 (BamHI site underlined), 59-CAGGTACATGAGCATCGTGAC-

CAC-39 and 59-CGGAATTCATGCTGCTTTTAGGACTCGTTCTGGCC-39

(EcoRI sites underlined), and 59-ACTTGTCACCCTTAAGCGTG-39 using

the pVL1392-Cr-P4H-1A vector generated above as a template. The pro-

ducts 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 CC125þ cells by electroporation at an electrical strength of

1375 V/cm in a transformation medium of 40 mM sucrose in water

(Shimogawara et al., 1998). 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 mg/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 transform-

ants were picked up and grown in TAP medium with Zeocin (10 mg/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 mL in size was resuspended in 50 mL of 10 mM Na-

EDTA by vortexing, incubated at 1008C for 5 min, vortexed, and centri-

fuged at 12,000g for 1 min. A 3-mL fraction of the supernatant was used as

a template in the PCR reactions. The primers 59-TTTAAGATGTTGAGT-

GACTTCTC-39 and 59-ACTTGTCACCCTTAAGCGTG-39 (primer pair 1 in

Figure 6B) were used to amplify a 750-bp fragment extending from the

upstream RBCS2 promoter to the 39 end of the sense strand of Cr-P4H-1,

and primers 59-GATTTAAATGCCAGAAGGAGC-39 and 59-CAGGTACAT-

GAGCATCGTGACCA-39 (primer pair 2 in Figure 6B) were used to amplify

a 950-bp fragment extending from the downstream RBCS2 promoter to

the 39 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 mm), 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, 2002) 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

�908C and gradually warmed to 08C. They were then removed from the

specimen carriers and infiltrated into Epon Ember 812 (Electron Micros-

copy Sciences) at room temperature and polymerized at 608C 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 cap-

tured 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 trans-

mission 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 Myllyla, 1982) at 308C. The Tris-

HCl buffer was replaced with 50 mM HEPES, pH 6.8, and the concen-

tration of FeSO4 was increased to 200 mM (Kaska et al., 1987). 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 1008C for

10 min, followed by rapid cooling before addition to the enzyme reaction

mixture. Km values were determined as described previously (Myllyharju

and Kivirikko, 1997). The partially hydroxylated (Pro-Pro-Gly)10 peptide

was purified from the reaction mixture and sequenced as described

previously (Hieta and Myllyharju, 2002).

Preparation of cell extracts from RNAi and control lines for P4H activity

assay was performed as described previously (Kaska et al., 1987). Briefly,

25 3 106 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 13 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 48C, 300 mM KCl, 20 mM

MgCl2, 0.05 mM DTT, and 0.1% Triton X-100). After incubation for 1 h at

48C, the solution was centrifuged at 15,000g for 20 min, and the super-

natants 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 suspend-

ing an equal number of cells in 1 mL of TAP medium containing 10 mg/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 Nissila and Liisa Aijala (University of Oulu) for their

expert technical assistance, Elizabeth Harris (Duke University, Durham,

Chlamydomonas Prolyl 4-Hydroxylases 267

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.

Received March 22, 2006; revised November 14, 2006; accepted

December 5, 2006; published January 12, 2007.

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Chlamydomonas Prolyl 4-Hydroxylases 269

DOI 10.1105/tpc.106.042739; originally published online January 12, 2007; 2007;19;256-269Plant Cell

Katriina Keskiaho, Reija Hieta, Raija Sormunen and Johanna MyllyharjuProper Cell Wall Assembly

Has Multiple Prolyl 4-Hydroxylases, One of Which Is Essential forChlamydomonas reinhardtii

 This information is current as of June 16, 2018

 

Supplemental Data /content/suppl/2007/01/12/tpc.106.042739.DC1.html

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