Sub-proteome analysis in the green flagellate alga Chlamydomonas reinhardtii

32 Journal of Basic Microbiology 2009, 49, 32–41

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Review Article

Sub-proteome analysis in the green flagellate alga Chlamydomonas reinhardtii

Volker Wagner*, Jens Boesger* and Maria Mittag

Institute of General Botany and Plant Physiology, Friedrich-Schiller-University Jena, 07743 Jena, Germany * Both authors have contributed equally

In the past years, research on the flagellate unicellular alga Chlamydomonas reinhardtii has

entered a new era based on the availability of its complete genome. Since this green alga can be

grown relatively easy in a short time-range, sufficient biological material is available to

efficiently establish biochemical purification procedures of sub-cellular fractions. Combined

with the available genome sequences, this paved the way to perform analysis of specific

sub-proteomes by mass spectrometry. In this review, several approaches that provided

comprehensive lists of components of certain sub-cellular compartments and their biological

relevance will be described. These include proteins of chloroplast ribosomes, of flagella, of the

eyespot as well as posttranslational and environmentally modified sub-proteomes. The power

of such proteome approaches lies in the identification of novel components and modifications

of a given sub-proteome that have not been discovered before. Information is usually gained at

a large scale and is very valuable to further understand biological processes of a given cellular

sub-compartment. But clearly the arduous task has then to be performed to further analyze

the function of specific proteins/genes by RNA interference technology, mutant analyses or

methods for identifying the protein interaction network within a sub-proteome.

Keywords: Chlamydomonas reinhardtii / Posttranslational Modifications / Sub-proteomes

Received: September 11, 2008; accepted: October 14, 2008

DOI 10.1002/jobm.200800292

Introduction*

The green unicellular alga Chlamydomonas reinhardtii,

whose nuclear, chloroplast and mitochondrial genomes

have been fully sequenced [1–3], has served for many

years as a model to study certain cellular processes and

organelles in depth. These include the biogenesis and

function of the chloroplast, its photosynthetic machin-

ery, the composition of flagella and the basal body for

their relevance to human diseases, metabolism, nutri-

ent acquisition as well as components of the light sig-

naling pathway and of its circadian clock [1, 4, 5]. A

schematic view of a C. reinhardtii cell is presented in

Fig. 1. C. reinhardtii offers experimental advantages that

come close to yeast while yeast cannot be used for

many of the processes in focus. It is a haplont, but plus

Correspondence: Maria Mittag, Institute of General Botany and Plant Physiology, Friedrich-Schiller-University Jena, 07743 Jena, Germany E-mail: [email protected] Phone: 0049-(0)3641-949-201

and minus gametes can be induced by starvation in the

light. Genetic crosses and tetrad analysis are possible.

The alga can be grown photo-, mixo- and heterotrophi-

cally (on acetate) allowing analysis of mutants that

would be lethal, if obligatory photoautotroph. Numer-

ous molecular biology methods have been developed

over the past years for this alga and several reporter

genes (e.g. green fluorescent protein, luciferase) as well

as strong promoters are available [4–6].

During the last few years, proteomics has been estab-

lished as a powerful tool for understanding various

biological problems in several organisms [7]. Although

proteins can be identified down to the femtomol and

even the attomol range with modern mass spectrome-

try, many regulatory proteins within a crude extract

are not abundant enough to be unambiguously identi-

fied by mass spectrometry. For this reason, enrichment

of such proteins is often a prerequisite for efficient

proteome analysis. This can be achieved, for example,

by isolating cellular sub-compartments or conducting

Journal of Basic Microbiology 2009, 49, 32–41 Sub-proteome analysis in Chlamydomonas reinhardtii 33


affinity purification in addition, such as for phospho-

proteins. All these procedures require that enough

biological material is available. C. reinhardtii is well

suited with regard to this aspect, because it is easy to

grow in high amounts in a relatively short time range.

Therefore, biochemical procedures to purify sub-

cellular compartments can be efficiently established.

Moreover, the presence of the entire genome sequences

[3] as well as more than 200,000 EST sequences that

have been assembled into ca. 10,000 unique cDNAs

[8, 9] represents an efficient platform for mass spec-

trometry analysis. In this review, several exemplary

sub-proteomes of C. reinhardtii that have been analyzed

in the past years will be described. The focus will be on

the biological processes that can be studied in a given

sub-proteome and on the identification of novel com-

ponents.

Figure 1. Schematic view of a Chlamydomonas cell (modified from [3]). C. reinhardtii is a ~10 µm green alga with multiple mito-chondria, two anterior flagella for motility and mating, and one large chloroplast. The two flagella rooted in basal bodies are shown with intraflagellar transport (IFT) particle arrays between the axoneme and the flagellar membrane. The U-shaped chloroplast is also shown along with thylakoid membranes, the region of its pyrenoid and the eyespot consisting usually of two layers of carotenoid rich lipid globuli. Moreover, the central nucleus and mitochondria are depicted.

Chloroplast ribosomal proteins [14, 15]

Proteomics has also become a powerful tool for the

investigation of chloroplast function [reviewed by 10].

Primary chloroplasts are thought to have evolved by

endosymbiosis of a photosynthetic unicellular prokary-

ote into a eukaryotic host. During this evolutionary

process many genes of the prokaryotic endosymbiont

have been translocated into the nuclear genome of the

host [reviewed by 11]. Plastid gene expression is, there-

fore, also under control of the nuclear genes. It is often

regulated at the translational level and involves the

dynamic interplay between ribosomes, translation fac-

tors and mRNA-protein complexes [12, 13]. To under-

stand how mRNAs are translated into functional pro-

teins, a detailed understanding of all the components

of chloroplast translation, including the ribosomes is

needed. Therefore, proteins of the small (30S) and large

(50S) subunits of the chloroplast 70S ribosome of C. rein-

hardtii were analyzed by a proteome approach [14, 15].

As shown in Fig. 1, this green alga contains one large

U-shaped chloroplast. Its ribosomes were isolated using

a postmitochondrial fraction gained from a protein

crude extract by centrifugation. By successive sucrose

gradient fractionation, the plastid 70S ribosomes were

first separated from the cellular 80S ribosomes, and

then the 70S ribosomes were dissociated into 30S and

50S subunits. These subunits were again separated by

sucrose gradient centrifugation and their proteins were

further used for mass spectrometry analysis [14, 15].

In the small subunit twenty proteins were identified,

including orthologs of Escherichia coli S proteins and a

homolog of spinach plastid-specific ribosomal protein-3

(PSRP-3). In addition, a novel S1 domain-containing

protein, PSRP-7, was found. Among the identified pro-

teins, S2, S3 and S5 are predominantly larger than their

E. coli or spinach counterparts, containing N-terminal

extensions (S2 and S5) or insertion sequences (S3) [14].

The additional proteins and domains may point out

novel functions for the regulation of chloroplast trans-

lation in C. reinhardtii. In the large subunit of the 70S

ribosome, twenty-seven orthologs of E. coli large subunit

proteins were identified, as well as an ortholog of the

spinach plastid-specific ribosomal protein-6. Again,

several of the large subunit proteins of C. reinhardtii had

short extension or insertion sequences, but overall the

large subunit proteins were very similar to those of

spinach chloroplast and E. coli [15]. Notably, two pro-

teins of 38 and 41 kDa, designated RAP38 and RAP41,

were identified from the 70S ribosome that were not

found in either of the ribosomal subunits. Phylogenetic

analysis revealed these two proteins as paralogs of spin-

34 V. Wagner et al. Journal of Basic Microbiology 2009, 49, 32–41


ach CSP41, a chloroplast RNA-binding protein with

endoribonuclease activity [15].

Thus, proteomic analyses of the chloroplast ri-

bosomes identified a significant number of chloroplast-

unique additional domains of ribosomal proteins that

expand upon a basic bacterial 70S-like composition. In

a recent study [74], cryo-electron microscopy and single-

particle reconstruction revealed that these novel struc-

tural additions are located at optimal positions on the

chloroplast ribosome for interaction with mRNAs dur-

ing translation initiation. Visualization of these chloro-

plast-unique structures on the ribosome, combined

with mRNA cross-linking, allowed to propose a model

for translation initiation in chloroplasts in which

chloroplast-unique ribosomal proteins interact with

plastid-specific translation factors and RNA elements to

facilitate regulated translation of chloroplast mRNAs

[74].

The composition of the flagellum [16]

Cilia and flagella, which are essentially identical, are

widespread cell organelles that have been highly con-

served throughout evolution and play important roles

in motility, sensory perception and the life cycles of

eukaryotes ranging from protists to humans. Defects in

cilia in humans can cause severe diseases such as poly-

cystic kidney disease (PKD), retinal degeneration, hy-

drocephalus, or changes in the left-right symmetry of

organs, collectively known as ciliopathies [17]. Al-

though C. reinhardtii and mammals are separated by

more than 109 years of evolution, C. reinhardtii flagella

are amazingly similar in structure and function to

mammalian motile cilia [18]. They are composed of

nine microtubular doublets surrounding two central

microtubular singlets. The axoneme of motile flagella

includes substructures such as dynein arms and radial

spokes that generate and control axoneme bending [19].

The flagellum also contains matrix proteins that are

not tightly associated with the flagellar membrane or

the axoneme. They serve diverse functions and can be

involved in intraflagellar transport (IFT) [20].

Proteome analysis of flagellar proteins from C. rein-

hardtii was undertaken with different sub-fractions of

this organelle [16]. Therefore, algal cells were first

deflagellated. The flagella were then sub-fractionated in

different parts, including membrane and axonemes,

membrane and matrix as well as a KCl extract of

axonemes and the remaining axonemes. By this way,

a comprehensive list of flagellar proteins along with

sub-cellular localization within the flagellum was

provided. All identified peptides along with the

corresponding proteins can be accessed via the web

site http://labs.umassmed.edu/chlamyfp/index.php. 360

proteins were identified by five or more peptides, and

additional 292 proteins by two to five peptides [16].

Among the identified proteins, many motor proteins

were present. Moreover, more than 90 putative signal

transduction proteins were found. These include, for

example, 21 protein kinases and 11 protein phosphata-

ses. These findings are consistent with the fact that a

large number of flagellar proteins are phosphorylated

[21], and with the roles of reversible protein phosphory-

lation in the control of flagellar motility [22], signaling

[23], and probably assembly [20]. Another functional

group of this category contained 27 EF hand-containing

proteins that are potential Ca2+-binding proteins, con-

sistent with the role of Ca2+ in the control of flagellar

behavior [24] and signaling [25, 26]. In addition to signal

transduction proteins, also proteins for nucleotide pro-

duction and metabolism were found, including en-

zymes of the late glycolytic pathway. Of note are also

homologues of vertebrate disease proteins that are

involved in PKD (e.g. homologues of polycystin2, fibro-

cystin, scorpion, quilin and three NIMA kinases), hy-

drocephalus (homologues of hydin and napa) or pro-

teins associated with male sterility in mouse (e.g.

homologues of “Parkin-coregulated gene” protein or of

a protein encoding a phosphatase1 catalytic subunit)

[16].

Proteins of the centriole [31]

Centrioles are composed of nine triplet microtubules

arranged in a cylinder [27]. During interphase, centri-

oles act as basal bodies by templating the formation of

cilia and flagella. Some human diseases such as the

Bardet-Biedl syndrome are known to result from de-

fects in proteins that are localized in the basal body

[28–30]. Biochemical purification of centrioles is subtle.

For this purpose, algal cells were first deflagellated and

then lysed in detergent. Basal bodies were enriched by

sucrose step gradients, followed by equilibrium cen-

trifugation in a continuous gradient of Nycodenz [31].

Proteomic analysis of this organelle revealed some pro-

teins already known to be localized in the basal body,

such as centrin, epsilon tubulin, and the cartwheel

protein BLD10p [31]. In addition, 27 so-called BUG

(basal-body proteins with up-regulated genes) proteins

were identified. Genes that are up-regulated during

flagellar assembly encode these. They are likely to be

directly involved in ciliogenesis. Moreover, 18 strong



candidate centriole proteins were found, designated

POC (proteome of centriole), which are not encoded by

genes up-regulated during flagellar regeneration [31]

and conserved in species with centrioles and/or in the

human centrosome. They are presumably core struc-

tural components of the centriole.

Analysis of the flagellar and basal body sub-

proteomes have provided an efficient basis to further

understand the biological structure and processes

within these cell organelles and notably, the knowledge

can be used to further gain insight into human

ciliopathies.

The eyespot and its components [33]

The eyespot apparatus of flagellate green algae is a

primordial visual system, which can detect both, light

direction and intensity and is thus important for the

phototactic orientation of these algae [reviewed in 32].

In C. reinhardtii, it is a singular structure usually com-

posed of two layers of highly ordered carotenoid-rich

lipid globuli that are situated at the periphery of the

chloroplast. Thylakoid membranes subtend these glob-

ule layers. Moreover, the outermost globule layer is

attached to specialized areas of the chloroplast enve-

lope membranes and the adjacent plasma membrane,

in which the photoreceptors are considered to be local-

ized. For purification of this complex ultrastructure,

flotation on successive sucrose gradients was applied

[33]. Thereby, a fraction enriched in the eyespot lipid

globuli and the associated parts of chloroplast and

plasma membranes was gained and used for its pro-

teomic analysis. 202 proteins of the eyespot apparatus

that were covered by at least two peptides per protein

were identified. These proteins included the already

known six proteins of the eyespot, EYE2 and MIN1, two

proteins that are relevant for eyespot assembly [re-

viewed in 34], two splicing variants of the retinal bind-

ing protein COP (Chlamydomonas opsin), and two unique

seven-transmembrane domain (TMD) photoreceptors,

COP3 and 4, which are better known as channelrho-

dopsins ChR-1 and ChR-2 [reviewed in 35]. The other

proteins include different functional categories. Some

are involved in carotenoid and retinal biosynthesis. One

group involving eight candidates contains a PAP fi-

brillin domain. Fibrillins were known as proteins of

carotenoid-rich fibrils and plastoglubules in chromo-

plasts and chloroplasts and play an important role in

carotenoid sequestration [36–38]. Yet another category

contains members of potential signal transduction

cascades such as Ca2+-binding proteins, kinases and

phosphatases and even potential chemotaxis-related

proteins [33]. The eyespot apparatus acquires light in-

formation via photoreceptors and forwards it through

signaling pathways to the flagella. In these signaling

cascades, Ca2+ is known to be involved [24, 39, 40]. In

the eyespot proteome, a significant number of thyla-

koid and chloroplast envelope related proteins were

also found. This can be expected since the carotenoid

lipid globule layers are subtented by thylakoid mem-

branes and overlaid by the chloroplast envelope mem-

branes. But one has to be careful to conclude that

known proteins of the chloroplast thylakoid have the

same localization and function in the eyespot. The al-

pha, beta and gamma subunits of the chloroplast ATP

synthase seem to have a specialized localization and

possibly function within the eyespot [41]. C. reinhardtii

proteome data from the eyespot have been deposited

on a protein network site (http://www2.uni-jena.de/

biologie/chlamy/index.php?page=search&cmd=search)

of C. reinhardtii.

Proteomic analysis of mitochondria from C. reinhardtii [43]

Like chloroplasts, mitochondria are of prokaryotic ori-

gin and have evolved by endosymbiosis. In C. reinhardtii,

mitochondria occupy about 1–3% of the cell volume of

photosynthetically grown algae, while the chloroplast

occupies about 40% [42]. A proteomic approach of mi-

tochondrial proteins from C. reinhardtii resulted in the

identification of proteins that are common among

strict anaerobic and microaerophilic prokaryotes, but

that are very rare in eukaryotes [43]. Thus, pyruvate

formate-lyase (PFL) that catalyzes the non-oxidative

conversion of pyruvate to formate and acetyl-CoA along

with its activating enzyme PFL-AE were found, showing

the existence of a PFL system in this photosynthetic

alga. Moreover, peptides from phosphotransacetylase

and acetate kinase were identified. The phosphotrans-

acetylase-acetate kinase pathway is a common route of

ATP synthesis or acetate assimilation among prokaryo-

tes, but not in eukaryotes. It is also remarkable that an

oxygen producing eukaryotic alga has a broad spectrum

of pyruvate-, ethanol- and acetate-metabolizing en-

zymes that were previously considered as specific to

anaerobic eukaryotic lineages [43]. However, this may

be necessary for the alga to adjust its metabolism to the

different environmental conditions to which it can be

exposed, including anaerobiosis, especially in darkness.

The natural habitats of C. reinhardtii are fresh water

ponds and soil. Notably, the first strain was isolated in



a potato field in Amherst, Massachusetts (USA) in 1945

and designated by G.M. Smith as isolate 137c [42].

Phosphoproteomes in C. reinhardtii

a. General considerations Many processes are regulated at the posttranslational

level. One of the key modifications of cellular signaling

processes represents phosphorylation. It can affect

proteins functions, activities, localization and interac-

tions. About one-third of all proteins are thought to be

phosphorylated [44]. The analysis of phosphorylated

proteins and the characterization of in vivo phosphory-

lation sites along with their relevant kinases under

different biological conditions are still considered to be

one of the most challenging tasks in current proteomic

research [45, 46]. This has several reasons. First, the

stoichiometry of phosphorylation is generally relatively

low; only a small fraction of the available intracellular

pool of a protein is phosphorylated at any given time as

a result of a stimulus. Second, the phosphorylated sites

on proteins might vary implying that any given phos-

phoprotein is heterogeneous (i.e. exists in several dif-

ferent phosphorylated forms). Third, many of the sig-

naling molecules are present at low abundance within

cells [47]. Connecting specific kinases with relevant

targets remains a further challenge [46].

Specific enrichment of phosphorylated proteins or

peptides is a prerequisite for efficient phosphoprotein

analysis. Immobilized metal-affinity chromatography

(IMAC) is a frequently applied technique for phos-

phopeptide and -protein enrichments [45]. Thereby,

phosphorylated peptides and proteins are bound to the

stationary phase with positively charged metal-ions

that are on their part bound to the column material.

Various metal-ions such as Fe3+, Ga3+, Al3+ or Zr3+ have

been used for selectivity and phosphopeptide recovery.

Especially Ga3+ ions have proven well in different stud-

ies [48–49]. Moreover, metal oxide affinity chromatog-

raphy (MOAC) using, for example, titanium dioxide has

been used to enrich phosphopeptides. Like the above-

mentioned metal ions, metal oxides preferentially in-

teract with phosphopeptides in comparison to unmodi-

fied peptides. In C. reinhardtii, both IMAC and MOAC

have been established [49–53].

b. Environmentally modulated phosphoproteomes of photosynthetic membranes [52, 53, 75, 76] The thylakoid membranes in chloroplasts of plants

and green algae represent the location for photosynthe-

sis. Several proteins of the thylakoid membrane that

make up photosystem II complex (PSII) and its light-

harvesting chlorophyll a/b binding proteins (LHCII)

antennae undergo light- and redox-dependent phos-

phorylation [54, 55]. Phosphorylation of LHCII controls

a process designated photosynthetic State transitions.

In State 1 more energy is transferred to PSII, whereas in

State 2 a proportion of the excitation energy is redis-

tributed to PSI [56–58]. To map in vivo phosphorylation

sites in photosynthetic membranes of C. reinhardtii un-

der different environmental conditions, the photosyn-

thetic membranes were isolated from the algal cells

and exposed to different conditions that affect photo-

synthesis [52]. These included (i) dark aerobic, corre-

sponding to photosynthetic State 1; (ii) dark under ni-

trogen atmosphere, corresponding to photosynthetic

State 2; (iii) moderate light and (iv) high light. The sur-

face-exposed phosphorylated peptides were cleaved

from the membrane by trypsin and finally enriched by

IMAC and analyzed by mass spectrometry. By this way,

19 in vivo phosphorylation sites belonging to 15 proteins

in C. reinhardtii were found [52]. The State 1- to State

2-transition induced phosphorylation of the PSII core

components D2 and PsbR and quadruple phosphoryla-

tion of a minor LHCII antennae subunit, CP29, as well

as phosphorylation of constituents of a major LHCII

complex, Lhcbm1 and Lhcbm10. Exposure of the cells

to either moderate or high light caused additional

phosphorylation of the D1 and CP43 proteins of the

PSII core. The high light treatment led to specific phos-

phorylation of CP29 at seven distinct residues, phos-

phorylation of another minor LHCII constituent, CP26

at a single threonine, and double phosphorylation of

additional subunits of a major LHC complex.

Mass spectrometry analysis of C. reinhardtii in the

State 1 and State 2 also revealed that the LHCII-like

CP29 protein was phosphorylated at Thr6, Thr16,

Thr32, and Ser102 in State 2, whereas in State

1-exposed cells only phosphorylation of Thr6 and Thr32

was detected. The LHCI-PSI supercomplex isolated from

the alga in State 2 was found to contain strongly associ-

ated CP29 in phosphorylated form. Electron microscopy

suggested the binding site for this highly phosphory-

lated CP29 in PSI and it was postulated that redox-

dependent multiple phosphorylation of CP29 in green

algae is an integral part of the State transition process

in which the structural changes of CP29, induced by

reversible phosphorylation, determine the affinity of

LHCII for either of the two photosystems [75].

Besides the phosphorylation sites detection, mass

spectrometry analyses of the surface-exposed peptides

cleaved by trypsin from the thylakoid membranes of

the green alga uncovered the N-termini of nuclear-



encoded chlorophyll a/b-binding proteins and their

N-terminal acetylation. The N-termini of mature chlo-

rophyll a/b-binding proteins differed from those earlier

predicted by bioinformatics [52, 76].

In another approach, C. reinhardtii was acclimated to

limiting environmental CO2 conditions. Under these

conditions, a CO2-concentrating mechanism (CCM) is

induced that is associated with transcriptional regula-

tion of many genes [59]. Thereby, specific protein phos-

phorylation at the surface of thylakoid membranes was

investigated and found to be redox-dependent [53]. One

phosphopeptide belonged to an unknown expressed

protein (UEP). Moreover, three phosphorylated peptides

of the Lci5 protein that is encoded by the low-CO2-

inducible gene 5 were identified.

c. The phosphoproteome of the eyespot [51] As mentioned before, proteomic analysis of the eyespot

of C. reinhardtii revealed the presence of a limited num-

ber of kinases and phosphatases [33], suggesting that

reversible protein phosphorylation might play a role in

the light-signaling cascade. To identify the targets of

the kinases and phosphatases, a functional phospho-

proteome approach was applied [51]. Since the proteins

of the eyespot have a rather hydrophobic character,

a specialized protocol involving digestion with the

endopeptidase LysC prior to trypsin was used for gener-

ating the phosphopeptides from the eyespot that were

enriched by IMAC. Proteins from the eyespot were dis-

solved in 4 M urea, in which LysC still has an activity of

86%. In total, 68 different phosphopeptides correspond-

ing to 32 known proteins of the eyespot along with 52

precise in vivo phosphorylation sites were identified by

mass spectrometry. Comparisons to other known phos-

phoproteins from C. reinhardtii obtained by phosphopro-

teomic approaches with whole cell extracts [49] or thy-

lakoid membranes [52, 53] revealed that only a few of

these phosphoproteins (6) were overlapping with the

eyespot phosphoproteome. Thus, a significant number

of phosphoproteins could only be identified when the

sub-cellular eyespot fraction was enriched prior to en-

richment of its phosphopeptides by IMAC. The identi-

fied phosphoproteins belong mainly to four functional

categories: carotenoid and fatty acid metabolism, (puta-

tive) light signaling pathway(s) and retina-related pro-

teins, thylakoid and chloroplast envelope-related pro-

teins and conserved and novel proteins of yet unknown

functions. Some phosphoproteins are clearly involved

in the light signaling pathway(s). These include the two

photoreceptors ChR-1 and ChR-2, which contain three

and one phosphorylation sites, respectively. These sites

are localized in a cytoplasmatic loop with close prox-

imity to the seven transmembrane domain channel-

forming regions. It is striking that the relative position

of the functional sites of phosphorylation is highly

conserved within the green algal and vertebrate rho-

dopsins, implying functional relevance for the regula-

tion of these unique directly light-gated channels [51].

In this context, it should be also pointed out that ChR-2

has been successfully expressed for light stimulation of

different systems including Caenorhabditis elegans and

mammalian neurons [60, 61]. It was even delivered to

retinal ganglion cells in a rodent model of inherited

blindness [62].

Two other examples of phosphoproteins that repre-

sent potential members of the light-signaling path-

way(s) within the eyespot include an EF hand contain-

ing Ca2+-binding protein and the SOUL3 heme-binding

protein [51]. It is known that extra-cellular calcium-

fluxes are intricately involved in the behavioral re-

sponses of C. reinhardtii to light [reviewed in 23] and

that both ChRs can conduct Ca2+ [63]. A SOUL heme-

binding protein was found in a screen for chicken

mRNAs specifically expressed in the retina and pineal

gland [64], indicating that certain proteins seem to be

indeed conserved with regard to their localization from

primitive visual algal systems to the highly sophisti-

cated visual system of animals.

Redox signaling in C. reinhardtii and the influence of Cadmium [65–67]

Pollution of the environment with heavy metals in soil

or water is often a result of industrial activity. Exposure

to Cadmium (Cd) can result in serious health problems.

Using proteomics along with comparative two-dimen-

sional gel electrophoresis, the effect of Cd on the so-

luble proteome of C. reinhardtii grown on acetate was

examined [65]. 20 proteins were found in a larger

amount compared to non-treated cells and 26 proteins

were decreased in the amount after Cd treatment. The

most drastic effect was the decrease in abundance of

both large and small subunits of the CO2 fixation en-

zyme, ribulose-1,5-bisphosphate carboxylase/oxygenase,

in correlation with several other enzymes involved in

photosynthesis, Calvin cycle and chlorophyll biosynthe-

sis. Other down regulated processes included fatty acid

biosynthesis as well as amino acid and protein biosyn-

thesis. On the other hand, proteins involved in glu-

tathione synthesis, ATP metabolism, response to oxida-

tive stress and protein folding were up-regulated in the

presence of Cd [65]. It was also found that many of the

Cd-sensitive proteins were regulated via two major



cellular thiol redox systems, thioredoxin and glutare-

doxin. Notably, targets of thioredoxins in C. reinhardtii

are known from a proteome approach [66]. Thereby, a

thioredoxin affinity approach was taken and 55 targets

were identified by mass spectrometry. Some of these

targets were known from higher plants and cyanobacte-

ria, but 26 were novel, providing an efficient platform

for further studying their functions and importance

within redox signaling. Recently, 25 in vivo targets of

S-thiolation were identified in C. reinhardtii [67]. Gluta-

thionylation is the major form of S-thiolation in cells.

This reversible redox post-translational modification

consists of the formation of a mixed disulfide between a

free thiol on a protein and a molecule of the tripeptide

glutathione. Several of the identified targets were relat-

ed to photosynthesis, such as the Calvin cycle enzymes

phosphoglycerate kinase and ribose-5-phosphate isom-

erase. A number of targets were also related to stress

responses, such as chaperones and peroxiredoxins [67].

Further proteome approaches in C. reinhardtii

There have been also other functional proteomic ap-

proaches in C. reinhardtii [68, 69]. A few further strate-

gies are shortly described below. For example, func-

tional proteomics was applied to find proteins that are

regulated by the circadian clock. Two circadian ex-

pressed proteins, a protein disulfide isomerase and a

tetratricopeptide repeat protein, which were enriched

by heparin affinity chromatography, were identified by

this way [70]. Shotgun proteomic profiling technologies

were applied to C. reinhardtii that detected 1069 proteins

of this green alga. These data were used to validate the

in silico gene models [71]. Co-immunoprecipitations with

antibodies directed against a specific protein were car-

ried out to precipitate interaction partners that can

then be analyzed by mass spectrometry. Such an effort

was undertaken, for example, to characterize the “in-

teractome” of the vesicle-inducing protein in plastids,

designated VIPP1 in C. reinhardtii [72].

Comparative quantitative proteomics is of high in-

terest to study variations in the protein pool of biologi-

cal processes that are induced under certain conditions.

Thereby, stable isotope labeling by amino acids in cell

culture can be used. Such an approach was undertaken

to investigate the remodeling of bioenergetic pathways

under iron deficiency in C. reinhardtii [73]. Proteins that

show a decreased or increased expression under iron

deficiency were identified. The data suggested that

iron-deprivation induces a transition from photohet-

erotrophic to a primarily heterotrophic metabolism.

Conclusions

This review has focused mainly on large-scale proteome

approaches that have been undertaken in the past years

with C. reinhardtii. The proteomes described in this re-

view underline the power of functional proteome ap-

proaches, where cellular compartments are purified

and the proteins are identified by mass spectrometry.

By this way, hundreds of proteins have been character-

ized that belong to specific sub-cellular compartments

of C. reinhardtii and/or that are modified at the post-

translational level or modulated by the environment.

These data have provided new insights into a variety of

biological processes in C. reinhardtii. They create an

efficient basis for the further functional characteriza-

tion of involved proteins and provide a platform for

knowledge transfer to other organisms including not

only plant, but also prokaryotic and animal systems.

Acknowledgements

We thank Carsten Milkowski for helpful comments on

the manuscript. Proteome analysis of C. reinhardtii

would not have reached such a level without the free

delivery of EST and genome sequences from the ge-

nome projects of C. reinhardtii in the USA (Department

of Energy, Joint Genome Institute) and Japan (Kazusa

DNA Research Institute). Our work was supported by

grants of the Deutsche Forschungsgemeinschaft.

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Sub-proteome analysis in the green flagellate alga Chlamydomonas reinhardtii