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Chlamydomonas reinhardtii produces a profilin with unusual biochemical properties

¹ Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA
² Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
³ Department of Disease and Stress Biology, John Innes Centre, Norwich, NR4 7UH, UK

View larger version (85K): [in a new window]   Fig. 1. Comparison of profilin amino acid sequences. (A) Multiple sequence alignment of the deduced amino acid sequence for CrPRF with plant, yeast, vertebrate and Vaccinia virus profilins. ClustalW analysis of the following sequences was performed using MacVector 7.0 software: Chlamydomonas reinhardtii (CrPRF; GenBank accession number AF335423), Arabidopsis thaliana profilin1 (AtPRF1; AAG10090), AtPRF2 (AAG10088), AtPRF3 (AAG10089), AtPRF4 (AAG10091), AtPFN4 (AAB39479), Zea mays profilin1 (ZmPRO1; X73279), ZmPRO2 (X73280), ZmPRO3 (X73281), ZmPRO4 (AF032370), ZmPRO5 (AF201459), Ricinus communis (RcPRO; AF092547), Schizosaccharomyces pombe (P39825), Saccharomyces cerevisiae (P07274), bovine profilin I (P02584), human profilin I (A28622) and Vaccinia virus profilin (P20844). Residues that are conserved in >51% of the displayed sequences are shown in bold and shaded grey. Gaps (-) were introduced to optimize the alignment. Conserved residues implicated in PLP binding are denoted by an asterisk, whereas those involved in actin binding are marked by a hash (#). The two regions of primary sequence that contribute to a plant-specific patch are overlined. Noteworthy substitutions that are predicted to affect CrPRF’s association with ligands are marked with a circle. (B) Phylogenetic comparison of the profilins shown in (A). The ClustalW multiple sequence alignment was analysed with a UPGMA algorithm and bootstrapped 1000 times using MacVector 7.0 software to create the tree shown here. Similar results were obtained using an neighbour-joining algorithm (not shown).

View larger version (32K): [in a new window]   Fig. 2. CrPRF is the only profilin-like gene in Chlamydomonas. (A) Southern blot analysis of 10 µg wild-type genomic DNA digested by BamHI (B), EcoRI (E), SalI (S) or XhoI (X) revealed fragments of the predicted sizes, suggestive of a single CrPRF gene. (B) Northern blot analysis of 10 µg poly(A)+ mRNA from wild-type cells (NR) or from wild-type cells collected 30 minutes after deflagellation (R). Notably, the message increased dramatically following deflagellation. The message for the Chlamydomonas CRY gene (Yang and Sale, 1998) was used as a loading control in all northern blots (not shown).

View larger version (26K): [in a new window]   Fig. 3. Western analysis reveals CrPRF in cytoplasmic and flagellar compartments. (A,B) Coomassie Blue staining (A) and anti-CrPRF immunoblot (B) comparing cytoplasmic and flagellar fractions. (A) Each lane contained 20 µg protein from cell body extract (C.B.ext), isolated flagella from wild-type cells (WTfla), isolated axonemes from wild-type flagella (WTaxo) and isolated flagella from pf14 cells (pf14fla). Molecular weight standard positions are shown on the left. (B) Lanes 3-6 are the corresponding western blots using the affinity-purified CrPRF antibody for detection. Lanes 1 and 2 were loaded with 5 ng and 1 ng purified, recombinant CrPRF. Notably, isolated flagellae contained a significant fraction of profilin (lanes 4 and 6). By contrast, axonemes contained very little profilin (lane 5). (C) Western blot comparing CrPRF in flagellar fractions. Notably, little CrPRF was found in the axoneme (Axo; lane 2). By contrast, nearly all of the flagellar CrPRF was detergent soluble and found in the membrane-matrix fraction (Memb./Matr.; lane 3).

View larger version (59K): [in a new window]   Fig. 4. Immunofluorescence localization of CrPRF and actin in Chlamydomonas vegetative cells (A-C,F) and mt+ gametes (D,E) reveals CrPRF localization in the cytoplasm and flagella. (A-C,E) Double labelled with anti-CrPRF (FITC) and anti-actin (Cy5) antibodies and imaged by confocal microscopy. (D-F) Single labelled and observed by wide-field fluorescence microscopy. CrPRF was relatively intensely stained throughout the cell body, including prominent staining in two CrPRF-enriched regions (A-E, arrowheads) located at the anterior end of the cells. These CrPRF-enriched regions were located at the base of the flagellae near the basal bodies and near the F-actin-containing fertilization tubule in mt+ gametes (E). The CrPRF-enriched spots were observed in all cells, irrespective of fixation condition, but staining was enhanced in cells first fixed by acetone (C). (Cells in A, B and E were first fixed in formaldehyde, cells in C and F were fixed in acetone and cells in D and F were fixed in paraformaldehyde followed by acetone.) Anti-CrPRF also stained flagellae. This is illustrated in (D) using wide-field fluorescence microscopy, and in comparison with the negative control image (F). Notably, flagellar staining was not illustrated in images A-C and E owing to the focal plane. For each image, cells selected were representative of the entire population. Scale bars, 5 µm.

View larger version (15K): [in a new window]   Fig. 5. CrPRF is an extremely stable profilin. The stability of CrPRF (circles), HPRO1 (triangles) and ZmPRO5 (diamonds) proteins was determined by incubating them with increasing concentrations of urea and measuring their intrinsic tryptophan fluorescences. The relative fluorescence was plotted against the urea concentration and fitted to a sigmoid curve. For this representative experiment, the denaturation midpoint for HPRO1 was 3.3 M, for ZmPRO5 4.0 M and for CrPRF 5.2 M.

View larger version (20K): [in a new window]   Fig. 6. CrPRF has a low affinity for PtdIns(4,5)P2. (A) Microfiltration of profilin-PtdIns(4,5)P2 complexes shows that little CrPRF bound to lipid micelles. The indicated concentrations of PtdIns(4,5)P2 in micelles were incubated with 2.5 µM profilin and spun through a 30,000 molecular weight cut-off filter. The flow through was analysed by SDS-PAGE. The 14-kDa region of gels from a representative experiment are shown for CrPRF, ZmPRO5 and HPRO1 (top). The intensity of each Coomassie-stained band was determined with a densitometer and normalized against the intensity of the profilin band found in the flow through in the absence of PtdIns(4,5)P2. Bars (bottom) represent the percentage of CrPRF (black), ZmPRO5 (grey) or HPRO1 (white) present in the flow through from two independent experiments. (B) The hydrolysis of PtdIns(4,5)P2 by phospholipase C (PIC) was measured in the absence or presence of 5 µM profilin. Each bar represents the average (± standard deviation) of at least four independent determinations. PIC activity in the absence of profilin (ø profilin) was set to 100%.

View larger version (19K): [in a new window]   Fig. 7. CrPRF has a high apparent affinity for maize pollen G-actin. A representative experiment shows that CrPRF shifts the steady state Cc (see below) for actin assembly. Increasing concentrations of pollen G-actin were polymerized alone (squares) or in the presence of 1 µM CrPRF (circles), 1 µM ZmPRO5 (diamonds) or 1 µM HPRO1 (triangles). The x-axis intercepts of each regression line (Cc values) were 0.29 µM in the absence of profilin and 0.74 µM, 0.71 µM and 0.91 µM in the presence of CrPRF, ZmPRO5 and HPRO1, respectively. The calculated apparent Kd values were 0.35 µM for CrPRF1, 0.40 µM for ZmPRO5 and 0.18 µM for HPRO1. Abbreviation: A.U., arbitrary light-scattering units.

View larger version (17K): [in a new window]   Fig. 8. CrPRF inhibits nucleotide exchange. (A) Representative experiments show that profilins from diverse organisms have substantially different effects on the initial rate of nucleotide exchange for G-actin. The incorporation of -ATP by 0.5 µM G-actin (RSMA) in low salt buffer alone (curve 3) or in the presence of 0.1 µM HPRO1 (curve 1), 2.5 µM ZmPRO5 (curve 2), 0.5 µM CrPRF (curve 4) or 2.5 µM CrPRF (curve 5) was monitored over time. The curves shown are fits of raw data (not shown) with a single exponential function. Human profilin I dramatically enhanced the initial rate of nucleotide exchange, whereas ZmPRO5 had little effect and CrPRF significantly decreased the initial rate. (B) The effect of a range of CrPRF concentrations (0.1-20 µM) on the initial rate of nucleotide exchange of 2.0 µM G-actin (maize pollen) in low ionic strength buffer is shown. Initial rates were determined by fitting the first 240 seconds of curves similar to those shown in (A) to a single exponential function. The initial rates were plotted against the concentration of CrPRF and fitted to the equation described in Methods. The calculated dissociation constant was 0.11 µM.

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