Protein In Chlamydomonas Reinhardtii Research Articles

Production and secretion of functional SARS-CoV-2 spike protein in Chlamydomonas reinhardtii.

The spike protein is the major protein on the surface of coronaviruses. It is therefore the prominent target of neutralizing antibodies and consequently the antigen of all currently admitted vaccines against SARS-CoV-2. Since it is a 1,273-amino acids glycoprotein with 22 N-linked glycans, the production of functional, full-length spike protein was limited to higher eukaryotes. Here we report the production of full-length SARS-CoV-2 spike protein – lacking the C-terminal membrane anchor – as a secreted protein in the prefusion-stabilized conformation in the unicellular green alga Chlamydomonas reinhardtii. We show that the spike protein is efficiently cleaved at the furin cleavage site during synthesis in the alga and that cleavage is abolished upon mutation of the multi-basic cleavage site. We could enrich the spike protein from culture medium by ammonium sulfate precipitation and demonstrate its functionality based on its interaction with recombinant ACE2 and ACE2 expressed on human 293T cells. Chlamydomonas reinhardtii is a GRAS organism that can be cultivated at low cost in simple media at a large scale, making it an attractive production platform for recombinant spike protein and other biopharmaceuticals in low-income countries.

Overexpression of native ORANGE (OR) and OR mutant protein in Chlamydomonas reinhardtii enhances carotenoid and ABA accumulation and increases resistance to abiotic stress

The carotenoid content of plants can be increased by overexpression of the regulatory protein ORANGE (OR) or a mutant variant known as the ‘golden SNP’. In the present study, a strong light-inducible promoter was used to overexpress either wild type CrOR (CrORWT) or a mutated CrOR (CrORHis) containing a single histidine substitution for a conserved arginine in the microalgae Chlamydomonas reinhardtii. Overexpression of CrORWT and CrORHis roughly doubled and tripled, respectively, the accumulation of several different carotenoids, including β-carotene, α-carotene, lutein and violaxanthin in C. reinhardtii and upregulated the transcript abundance of nearly all relevant carotenoid biosynthetic genes. In addition, microscopic analysis revealed that the OR transgenic cells were larger than control cells and exhibited larger chloroplasts with a disrupted morphology. Moreover, both CrORWT and CrORHis cell lines showed increased tolerance to salt and paraquat stress. The levels of endogenous phytohormone abscisic acid (ABA) were also increased in CrORWT and CrORHis lines, not only in normal growth conditions but also in growth medium supplemented with salt and paraquat. Together these results offer new insights regarding the role of the native OR protein in regulating carotenoid biosynthesis and the accumulation of several carotenoids in microalgae, and establish a new functional role for OR to modulate oxidative stress tolerance potentially mediated by ABA.

Expression and characterization of human lactoferrin with tandem zinc finger protein in Chlamydomonas reinhardtii

Lactoferrin (LF) is a naturally occurring iron-binding glycoprotein with a number of biological functions. However, its bioavailability remains a major limitation for clinical applications. In this study, we explored the expression of functional human lactoferrin (hLF) containing tandem zinc finger protein (ZFP) repeats in Chlamydomonas reinhardtii. Codon-optimized ZFPn-hLF fusion genes, where n donates 0, 1, 2, or 3 repeats of ZFP, were transformed into C. reinhardtii CW-15 strain. A total of 23 ZFPn-hLF-expressing clones were obtained. The expression levels of the ZFPn-hLF proteins ranged from 1.97% to 2.32% of the total soluble protein. The expression of ZFPn-hLF proteins did not compromise the proliferation of C. reinhardtii. The recombinant ZFPn-hLFs retained the antibacterial activities toward Escherichia coli and Klebsiella variicola IV-3, and also the antioxidant activities toward DPPH and hydroxyl free radicals. ZFP fusion did not compromise the stability of hLF and enabled efficient internalization of hLF into normal human small intestine cells. Efficiency of the internalization was found to be dependent on the number of tandem ZFPs, incubation time and incubation temperature. Our results indicate that recombinant ZFPn-hLFs expressed in C. reinhardtii promotes the absorption of lactoferrin in human intestinal epithelial cells, and thus provide an effective strategy to enhance the bioavailability of lactoferrin for its utilization in clinical applications.

Nuclear genome shuffling significantly increases production of chloroplast-based recombinant protein in Chlamydomonas reinhardtii

Algae biotechnology holds great promise for the production of sustainable and environmentally friendly products, however more robust strains of microalgae must be developed before algae can be used as a commercially relevant production platform. Here we demonstrate a timely and effective approach to significantly improve a desired phenotype in algae through a combination of mutagenesis and genome shuffling. In this study, we engineered a strain of Chlamydomonas reinhardtii to produce recombinant green fluorescent protein in the chloroplast and then subjected the nuclear genome to various rounds of UV-induced mutagenesis and genome shuffling via sexual recombination, followed by isolation of strains with enhanced recombinant protein expression using fluorescence-activated cell sorting (FACS). The greatest success came from first UV-mutagenizing the host strain to create mutations in the nuclear genome, followed by FACS to isolate the best GFP-expressing mutant, then shuffling the nuclear genome of that mutant by mating it with selected wild-type strains, and then again isolating the best progeny via FACS. Using this strategy, a novel strain was evolved in <3 months to express GFP over 2% total soluble protein (TSP), a 15-fold increase from the initial recombinant strain which expressed GFP at 0.14% TSP. This significant increase in GFP expression from the chloroplast was accomplished without altering the GFP gene itself or reducing the growth rate of the strain, demonstrating the effectiveness of this strategy to enhance a specific phenotype and significantly improve product production within an algal species in a commercially relevant timeframe.

Building the GreenCut2 suite of proteins to unmask photosynthetic function and regulation.

The suite of GreenCut proteins, initially assembled in 2007 and updated in 2011 (GreenCut2), comprises 597 Chlamydomonas reinhardtii proteins; these proteins, identified as putative orthologues in all green lineage organisms examined, but not (or poorly conserved) in non-photosynthetic organisms, are potentially enriched for proteins affiliated with photosynthesis. The annotation of GreenCut2 proteins and the characterization of mutants with lesions in genes encoding those proteins identified catalytic components of the photosynthetic apparatus that were previously uncharacterized, as well as polypeptides likely associated with chloroplast biogenesis and potential regulatory factors and activities that link environmental conditions to dynamic control of photosynthetic activities. Analyses of strains devoid of specific GreenCut2 proteins are being aided by a genome-wide library of mutants for which the lesions are mapped, indexed and readily available to the community (https://www.chlamylibrary.org/). In this review we briefly include some milestones in the history of photosynthesis, explain the way in which the GreenCut protein assemblage was generated and describe potential functions of individual member proteins, especially those linked to photosynthesis.

Absolute quantification of selected photosynthetic electron transfer proteins in Chlamydomonas reinhardtii in the presence and absence of oxygen.

The absolute amount of plastocyanin (PC), ferredoxin-NADP+-oxidoreductase (FNR), hydrogenase (HYDA1), and ferredoxin 5 (FDX5) were quantified in aerobic and anaerobic Chlamydomonas reinhardtii whole cells using purified (recombinant) proteins as internal standards in a mass spectrometric approach. Quantified protein amounts were related to the estimated amount of PSI. The ratios of PC to FNR to HYDA1 to FDX5 in aerobic cells were determined to be 1.4:1.2:0.003:0. In anaerobic cells, the ratios changed to 1.1:1.3:0.019:0.027 (PC:FNR:HYDA1:FDX5). Employing sodium dithionite and methyl viologen as electron donors, the specific activity of hydrogenase in whole cells was calculated to be 382 ± 96.5 μmolH2min-1mg-1. Importantly, these data reveal an about 70-fold lower abundance of HYDA1 compared to FNR. Despite this great disproportion between both proteins, which might further enhance the competition for electrons, the alga is capable of hydrogen production under anaerobic conditions, thus pointing to an efficient channeling mechanism of electrons from FDX1 to the HYDA1.

Deletion of the chloroplast LTD protein impedes LHCI import and PSI-LHCI assembly in Chlamydomonas reinhardtii.

Nuclear-encoded light-harvesting chlorophyll- and carotenoid-binding proteins (LHCPs) are imported into the chloroplast and transported across the stroma to thylakoid membrane assembly sites by the chloroplast signal recognition particle (CpSRP) pathway. The LHCP translocation defect (LTD) protein is essential for the delivery of imported LHCPs to the CpSRP pathway in Arabidopsis. However, the function of the LTD protein in Chlamydomonas reinhardtii has not been investigated. Here, we generated a C. reinhardtii ltd (Crltd) knockout mutant by using CRISPR-Cas9, a new target-specific knockout technology. The Crltd1 mutant showed a low chlorophyll content per cell with an unusual increase in appressed thylakoid membranes and enlarged cytosolic vacuoles. Profiling of thylakoid membrane proteins in the Crltd1 mutant showed a more severe reduction in the levels of photosystem I (PSI) core proteins and absence of functional LHCI compared with those of photosystem II, resulting in a much smaller PSI pool size and diminished chlorophyll antenna size. The lack of CrLTD did not prevent photoautotrophic growth of the cells. These results are substantially different from those for Arabidopsis ltd null mutant, indicating LTD function in LHCP delivery and PSI assembly may not be as stringent in C. reinhardtii as it is in higher plants.

Heterologous expression of the N-acetylglucosaminyltransferase I dictates a reinvestigation of the N-glycosylation pathway in Chlamydomonas reinhardtii

Eukaryotic N-glycosylation pathways are dependent of N-acetylglucosaminyltransferase I (GnTI), a key glycosyltransferase opening the door to the formation of complex-type N-glycans by transferring a N-acetylglucosamine residue onto the Man5GlcNAc2 intermediate. In contrast, glycans N-linked to Chlamydomonas reinhardtii proteins arise from a GnTI-independent Golgi processing of oligomannosides giving rise to Man5GlcNAc2 substituted eventually with one or two xylose(s). Here, complementation of C. reinhardtii with heterologous GnTI was investigated by expression of GnTI cDNAs originated from Arabidopsis and the diatom Phaeodactylum tricornutum. No modification of the N-glycans was observed in the GnTI transformed cells. Consequently, the structure of the Man5GlcNAc2 synthesized by C. reinhardtii was reinvestigated. Mass spectrometry analyses combined with enzyme sequencing showed that C. reinhardtii proteins carry linear Man5GlcNAc2 instead of the branched structure usually found in eukaryotes. Moreover, characterization of the lipid-linked oligosaccharide precursor demonstrated that C. reinhardtii exhibit a Glc3Man5GlcNAc2 dolichol pyrophosphate precursor. We propose that this precursor is then trimmed into a linear Man5GlcNAc2 that is not substrate for GnTI. Furthermore, cells expressing GnTI exhibited an altered phenotype with large vacuoles, increase of ROS production and accumulation of starch granules, suggesting the activation of stress responses likely due to the perturbation of the Golgi apparatus.

Identification of Reference and Biomarker Proteins in Chlamydomonas reinhardtii Cultured under Different Stress Conditions.

Reference proteins and biomarkers are important for the quantitative evaluation of protein abundance. Chlamydomonas reinhardtii was grown under five stress conditions (dark, cold, heat, salt, and glucose supplementation), and the OD750 and total protein contents were evaluated on days 0, 1, 2, 4, and 6 of culture. Antibodies for 20 candidate proteins were generated, and the protein expression patterns were examined by western blotting. Reference protein(s) for each treatment were identified by calculating the Pearson’s correlation coefficient (PCC) between target protein abundance and total protein content. Histone H3, beta tubulin 1 (TUB-1), ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RBCL), and mitochondrial F1F0 ATP synthase subunit 6 (ATPs-6) were the top reference proteins, because they were expressed stably under multiple stress conditions. The average relative-fold change (ARF) value of each protein was calculated to identify biomarkers. Heat shock protein 90B (HSP90B), flagellar associated protein (FAP127) and ATP synthase CF0 A subunit (ATPs-A) were suitable biomarkers for multiple treatments, while receptor of activated protein kinase C1 (RCK1), biotin carboxylase (BCR1), mitochondrial phosphate carrier protein (MPC1), and rubisco large subunit N-methyltransferase (RMT1) were suitable biomarkers for the dark, cold, heat, and glucose treatments, respectively.

IFT trains in different stages of assembly queue at the ciliary base for consecutive release into the cilium.

Intraflagellar transport (IFT) trains, multimegadalton assemblies of IFT proteins and motors, traffic proteins in cilia. To study how trains assemble, we employed fluorescence protein-tagged IFT proteins in Chlamydomonas reinhardtii. IFT-A and motor proteins are recruited from the cell body to the basal body pool, assembled into trains, move through the cilium, and disperse back into the cell body. In contrast to this 'open' system, IFT-B proteins from retrograde trains reenter the pool and a portion is reused directly in anterograde trains indicating a 'semi-open' system. Similar IFT systems were also observed in Tetrahymena thermophila and IMCD3 cells. FRAP analysis indicated that IFT proteins and motors of a given train are sequentially recruited to the basal bodies. IFT dynein and tubulin cargoes are loaded briefly before the trains depart. We conclude that the pool contains IFT trains in multiple stages of assembly queuing for successive release into the cilium upon completion.

High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii.

SummaryMicroalga‐based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post‐translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C‐terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n, wherein n = 10 or 20]. The yields of the (SP)n‐fused Venus were higher than Venus without the glycomodule by up to 12‐fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus‐(SP)n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP)n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae‐based recombinant proteins.

Cell-free reconstitution reveals centriole cartwheel assembly mechanisms

How cellular organelles assemble is a fundamental question in biology. The centriole organelle organizes around a nine-fold symmetrical cartwheel structure typically ∼100 nm high comprising a stack of rings that each accommodates nine homodimers of SAS-6 proteins. Whether nine-fold symmetrical ring-like assemblies of SAS-6 proteins harbour more peripheral cartwheel elements is unclear. Furthermore, the mechanisms governing ring stacking are not known. Here we develop a cell-free reconstitution system for core cartwheel assembly. Using cryo-electron tomography, we uncover that the Chlamydomonas reinhardtii proteins CrSAS-6 and Bld10p together drive assembly of the core cartwheel. Moreover, we discover that CrSAS-6 possesses autonomous properties that ensure self-organized ring stacking. Mathematical fitting of reconstituted cartwheel height distribution suggests a mechanism whereby preferential addition of pairs of SAS-6 rings governs cartwheel growth. In conclusion, we have developed a cell-free reconstitution system that reveals fundamental assembly principles at the root of centriole biogenesis.

Iron deficiency cause changes in photochemistry, thylakoid organization, and accumulation of photosystem II proteins in Chlamydomonas reinhardtii.

A trace element, iron (Fe) plays a pivotal role in photosynthesis process which in turn mediates the plant growth and productivity. Here, we have focused majorly on the photochemistry of photosystem (PS) II, abundance of proteins, and organization of supercomplexes of thylakoids from Fe-depleted cells in Chlamydomonas reinhardtii. Confocal pictures show that the cell's size has been reduced and formed rosette-shaped palmelloids; however, there is no cell death. Further, the PSII photochemistry was reduced remarkably. Further, the photosynthetic efficiency analyzer data revealed that both donor and acceptor side of PSII were equally damaged. Additionally, the room-temperature emission spectra showed the fluorescence emission maxima increased due to impaired energy transfer from PSII to PSI. Furthermore, the protein data reveal that most of the proteins of reaction center and light-harvesting antenna were reduced in Fe-depleted cells. Additionally, the supercomplexes of PSI and PSII were destabilized from thylakoids under Fe-deficient condition showing that Fe is an important element in photosynthesis mechanism.

TEF30 Interacts with Photosystem II Monomers and Is Involved in the Repair of Photodamaged Photosystem II in Chlamydomonas reinhardtii.

The remarkable capability of photosystem II (PSII) to oxidize water comes along with its vulnerability to oxidative damage. Accordingly, organisms harboring PSII have developed strategies to protect PSII from oxidative damage and to repair damaged PSII. Here, we report on the characterization of the THYLAKOID ENRICHED FRACTION30 (TEF30) protein in Chlamydomonas reinhardtii, which is conserved in the green lineage and induced by high light. Fractionation studies revealed that TEF30 is associated with the stromal side of thylakoid membranes. By using blue native/Deriphat-polyacrylamide gel electrophoresis, sucrose density gradients, and isolated PSII particles, we found TEF30 to quantitatively interact with monomeric PSII complexes. Electron microscopy images revealed significantly reduced thylakoid membrane stacking in TEF30-underexpressing cells when compared with control cells. Biophysical and immunological data point to an impaired PSII repair cycle in TEF30-underexpressing cells and a reduced ability to form PSII supercomplexes after high-light exposure. Taken together, our data suggest potential roles for TEF30 in facilitating the incorporation of a new D1 protein and/or the reintegration of CP43 into repaired PSII monomers, protecting repaired PSII monomers from undergoing repeated repair cycles or facilitating the migration of repaired PSII monomers back to stacked regions for supercomplex reassembly.

Metabolic labeling with stable isotope nitrogen (15N) to follow amino acid and protein turnover of three plastid proteins in Chlamydomonas reinhardtii

BackgroundThe length of time that a protein remains available to perform its function is significantly influenced by its turnover rate. Knowing the turnover rate of proteins involved in different processes is important to determining how long a function might progress even when the stimulus has been removed and no further synthesis of the particular proteins occurs. In this article, we describe the use of 15N-metabolic labeling coupled to GC-MS to follow the turnover of free amino acids and LC-MS/MS to identify and LC-MS to follow the turnover of specific proteins in Chlamydomonas reinhardtii.ResultsTo achieve the metabolic labeling, the growth medium was formulated with standard Tris acetate phosphate medium (TAP) in which14NH4Cl was replaced with 15NH415NO3 and (14NH4)6Mo7O24.4H2O was replaced with Na2MoO4.2H2O. This medium designated 15N-TAP allowed CC-125 algal cells to grow normally. Mass isotopic distribution revealed successful 15N incorporation into 13 amino acids with approximately 98% labeling efficiency. Tryptic digestion of the 55 kDa SDS-PAGE bands from 14N- and 15N-labeled crude algal protein extracts followed by LC-MS/MS resulted in the identification of 27 proteins. Of these, five displayed peptide sequence confidence levels greater than 95% and protein sequence coverage greater than 25%. These proteins were the RuBisCo large subunit, ATP synthase CF1 alpha and beta subunits, the mitochondrial protein (F1F0 ATP synthase) and the cytosolic protein (S-adenosyl homocysteine hydroxylase). These proteins were present in both labeled and unlabeled samples. Once the newly synthesized 15N-labeled free amino acids and proteins obtained maximum incorporation of the 15N-label, turnover rates were determined after transfer of cells into 14N-TAP medium. The t½ values were determined for the three plastid proteins (RuBisCo, ATP synthase CF1 alpha and beta) by following the reduction of the 15N-fractional abundance over time.ConclusionWe describe a more rapid and non-radioactive method to measure free amino acid and protein turnover. Our approach is applicable for determination of protein turnover for various proteins, which will lead to a better understanding of the relationship between protein lifetime and functionality.

A Study of Functional Genomics for Unknown Proteins in Chlamydomonas reinhardtii

reinhardtii is a unicellular green alga, which has been used as a reference organism for identifying proteins. Five hundred hypothetical proteins in Chlamydomonas reinhardtii have been sequenced for knowing functions of the proteins in their families. Functions of Five hundred hypothetical proteins in Chlamydomonas reinhardtii were predicted using bioinformatics web tools. The web tools like CDD-BLAST, INTERPROSCAN and PFAM were used for the prediction of functions in the proteins by searching protein databases for the presence of conserved domains. The current study was useful in better knowing the functions of hypothetical proteins in Chlamydomonas reinhardtii and the various regulatory mechanisms, which interact to yield a final output from the system.

The GreenCut: re-evaluation of physiological role of previously studied proteins and potential novel protein functions

Based on comparative genomics, a list of proteins present in the green algal, flowering and nonflowering plant lineages, but not detected in nonphotosynthetic organisms, was assembled (Merchant et al., Science 318:245-250, 2007; Karpowicz et al., J Biol Chem 286:21427-21439, 2011). This protein grouping, previously designated the GreenCut, was established using stringent comparative genomic criteria; they are those Chlamydomonas reinhardtii proteins with orthologs in Arabidopsis thaliana, Physcomitrella patens, Oryza sativa, Populus tricocarpa and at least one of the three Ostreococcus species with fully sequenced genomes, but not in bacteria, yeast, fungi or mammals. Many GreenCut proteins are also present in red algae and diatoms and a subset of 189 have been identified as encoded on nearly all cyanobacterial genomes. Of the current GreenCut proteins (597 in total), approximately half have been studied previously. The functions or activities of a number of these proteins have been deduced from phenotypic analyses of mutants (defective for genes encoding specific GreenCut proteins) of A. thaliana, and in many cases the assigned functions do not exist in C. reinhardtii. Therefore, precise physiological functions of several previously studied GreenCut proteins are still not clear. The GreenCut also contains a number of proteins with certain conserved domains. Three of the most highly conserved domains are the FK506 binding, cyclophilin and PAP fibrillin domains; most members of these gene families are not well characterized. In general, our analysis of the GreenCut indicates that many processes critical to green lineage organisms remain unstudied or poorly characterized. We have begun to examine the functions of some GreenCut proteins in detail. For example, our work on the CPLD38 protein has demonstrated that it has an essential role in photosynthetic function and the stability of the cytochrome b 6 f complex.

Novel FixL homologues in Chlamydomonas reinhardtii bind heme and O2

Genome inspection revealed nine putative heme-binding, FixL-homologous proteins in Chlamydomonas reinhardtii. The heme-binding domains from two of these proteins, FXL1 and FXL5 were cloned, expressed in Escherichia coli, purified and characterized. The recombinant FXL1 and FXL5 domains stained positively for heme, while mutations in the putative ligand-binding histidine FXL1-H200S and FXL5-H200S resulted in loss of heme binding. The FXL1 and FXL5 [Fe(II), bound O2] had Soret absorption maxima around 415nm, and weaker absorptions at longer wavelengths, in concurrence with the literature. Ligand-binding measurements showed that FXL1 and FXL5 bind O2 with moderate affinity, 135 and 222μM, respectively. This suggests that Chlamydomonas may use the FXL proteins in O2-sensing mechanisms analogous to that reported in nitrogen-fixing bacteria to regulate gene expression.

Effects of rare codon clusters on the expression of a high-turnover chloroplast protein in Chlamydomonas reinhardtii

Expression of foreign proteins in chloroplasts has become an important field of plant genetic engineering. Optimized codon usage is generally thought to increase translational efficiency, but high speed translation of codon bias-adjusted mRNAs can also result in protein misfolding due to a lack of rare codons. In order to analyze the effect of rare codons on a native chloroplast protein in vivo, we modified the D1 subunit of photosystem II by fusing small peptides with different codons into a loop region which tolerates insertions without loss of function. Because of its high-turnover properties, the D1 protein represents an excellent test object to investigate the impact of rare codons on its translation. We choose codons for amino acids Arg, Leu, Ser, Ala and Gly which are rarely used and compared translation of the modified D1 proteins with the respective mutant proteins containing insertions with frequently used codons. Our data indicate that only rare Arg codons drastically affect synthesis of the D1 protein and cluster of rare Ser-codon can induce strategic ribosomal pausing sites.

SUMO (small‐ubiquitin‐like modifier) in subcellular fractions of Chlamydomonas reinhardtii reveal potential function

SUMO (Small ubiquitin‐like modifier), is a member of the family of ubiquitin‐like polypeptides that become covalently attached to intracellular target proteins. SUMOylation can alter the function, location, and/or half‐life of target proteins. Here we describe three SUMO proteins and three SUMO‐like proteins in Chlamydomonas reinhardtii. The three SUMO genes show differential RNA expression depending on environmental conditions. Western analysis also shows a shift in proteins recognized by the Chlamydomonas reinhardtii SUMO antibodies in response to changes in environmental conditions. The three SUMO‐like proteins are novel fusion proteins with C‐terminal SUMO. Chlamydomonas reinhardtii subcellular fractionation reveals SUMO proteins are conjugated to proteins involved in various cellular processes. Proposed SUMOylated target proteins can be resolved by one‐dimensional and two‐dimensional western analysis.Supported by NSF and the Nebraska Research Council.