1. Volume 6, Issue 3, Pages 931-944 (May 2013)
The Heme-Binding Protein SOUL3 of Chlamydomonas reinhardtii Influences Size and Position of the Eyespot 
Thomas Schulze, Sandra Schreiber, Dobromir Iliev, Jens Boesger, Jessica Trippens, Georg Kreimer, Maria Mittag 
Molecular Plant 
Volume 6, Issue 3, Pages (May 2013)
DOI: /mp/sss137
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
Heterologous Expression of SOUL3 in E. coli and Hemin-Binding Studies.
(A) Structure of SOUL3 in C. reinhardtii. The location of the SOUL/HBP and an NTF2-like domain are shown as well as the region (amino acids, aa, 234–421) used for antibody production.
(B) Full-length His-tagged SOUL3 was expressed in E. coli and purified using affinity chromatography (see the Methods section). The His-tag was removed with TAGzyme (see the Methods section). 10 µl of protein from each fraction were separated by 10% SDS–PAGE and used for immunoblotting with anti-SOUL3 antibody. CE, crude extract; FT, flow-through; W, wash step; E, eluate.
(C) Remaining His-tagged SOUL3 as well as the TAGzyme dipeptidase were removed by ion metal affinity chromatography. Approximately 60 µg of the tag-less purified SOUL3 protein (PP) were loaded on 100 µl hemin–agarose slurry and washed several times (W; see the Methods section). The eluate (E) was collected in 2X sample buffer. Again, 10 µl of each fraction were used as described in (B).
(D) In a saturation assay, six aliquots, of 0.1 nmol tag-less SOUL3 protein, were incubated with 0, 0.1, 0.25, 0.5, 1, and 10 nmol hemin, respectively, and loaded on 200 nmol (~50 µl) hemin–agarose. From each aliquot treated in this manner, 10 µl of the flow-through (FT) as well as the eluate (E) were loaded on a 10% SDS–PAGE and immunoblotted with anti-SOUL3 antibody.
(E) Intensities of SOUL3 protein bands were measured with ImageMaster 2D Elite version 4.01 (GE Healthcare). The percentage of SOUL3 present in the eluate was determined as the quotient to the sum of the flow-through and eluate SOUL3 signal strength, assuming that both represent the whole SOUL3 amount loaded on the hemin–agarose, and blotted against the total amount of free hemin added to the protein.
Molecular Plant 2013 6, DOI: ( /mp/sss137)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
Diurnal Expression Pattern of SOUL3 in C. reinhardtii and Complex Formation.
(A) Cells were grown under a light–dark cycle (LD 12:12) and harvested every 4 h. An asterisk indicates the beginning of the next light period at LD2. Protein cell extracts were prepared and 50 µg of protein per lane were separated by 10% SDS–PAGE and immunoblotted with anti-SOUL3 antibody (SOUL3) and an anti-CK1 antibody (CK1), respectively. To control for equal amounts of proteins loaded, the nitrocellulose membrane was Ponceau-stained before the immunological detection. From this stain, selected, unspecified protein bands are shown (LC).
(B) Quantification of SOUL3 and CK1 protein levels using ImageMaster 2D Elite version 4.01 (GE Healthcare). The highest level of protein for each replicate (CK1, n = 3; SOUL3, n = 4) was set to 100%. In the case of CK1, LD18 represented the highest level in all three cases, therefore lacking error bars. The one-way ANOVA test (GraphPad Prism) showed a significant difference for the CK1 expression levels (P < ) and no significant difference for SOUL3 expression levels (P = ) within the LD cycle.
(C) Cells were entrained by a LD cycle and released under constant dim light (LL0). At subjective day (LL28) and subjective night (LL38), they were harvested and used for crude extract preparations. Equal amounts of protein were separated on a linear 6–14% sucrose density gradient as described (Zhao et al., 2004). Fractions were collected; numbering starts with the high percentage (14%). 40 µl protein from the different fractions were separated by 12% SDS–PAGE and used for immunoblotting with anti-SOUL3 antibody.
Molecular Plant 2013 6, DOI: ( /mp/sss137)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
Localization Studies of SOUL3 in C. reinhardtii.(A) Different fractions containing total protein (TP), soluble protein (SP), peripheral membrane proteins (PM), and transmembrane proteins (TM) were prepared from C. reinhardtii SAG wild-type cells (see the Methods section). 50 µg protein of each fraction were separated by 10% SDS–PAGE and immunoblotted with anti-SOUL3 and anti-CYTf (lower panel) antibodies as a control for the TM fraction.
(B) Different protein fractions were isolated (see the Methods section). 4 µg proteins from each fraction along with a crude extract (CE) were separated by 11% SDS–PAGE and immunoblotted with anti-SOUL3. As a control for the different protein fractions, we used anti-ChR1 for the eyespot (EYE), anti-β-Tubulin (TUB) for the flagella (F), anti-Rubisco large subunit (RbcL) for stroma (S), and anti-CYTf as control for a TM protein found in chloroplast membranes (CM).
(C) Schematic view of an eyespot cross-section (modified from Schmidt et al., 2006). The eyespot consists of two carotenoid-rich globule layers (asterisk), which are each subtended by a thylakoid layer (black arrowheads). These globule layers are also attached to specialized areas of the outer chloroplast membranes (long arrow) and plasma membrane, where the photoreceptors ChR1 and ChR2 (unfilled arrowheads) are situated (reviewed in Hegemann, 2008). The dotted line represents the D4-rootlet reaching to the eyespot (Melkonian and Robeneck, 1984).
(D) Immunofluorescence micrographs of a group of three wild-type cells (upper panel) and a single wild-type cell (middle left panel) that was also pictured by bright field microscopy (middle right panel) with SOUL3 staining (magenta color). An asterisk labels the position of the pyrenoid. The lower panel shows an overlay of a wild-type cell with stained acetylated-α-Tubulin (green color) as well as stained SOUL3 (magenta color, left panel) and ChR1 (magenta color, right panel), respectively. Scale bars = 5 µm.
(E) Immunofluorescence micrograph of a wild-type cell labeled with preformed soluble immune complexes of anti-SOUL3/anti-rabbit coupled to Alexa555 (magenta color) and anti-ChR1/anti-rabbit coupled to Alexa488 (green color) (see the Methods section). Scale bar = 5 µm. The small panel shows a close-up view (magnification 3.2×) of the eyespot region.
Molecular Plant 2013 6, DOI: ( /mp/sss137)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
SOUL3 Shows a Different Localization Pattern in eye3-2 Mutant Cells.
(A) Crude extracts of strain 137c and mutant strain eye3-2 were prepared. 100 µg protein were separated by 10% SDS–PAGE along with molecular mass standards and immunoblotted with anti-SOUL3 antibody. To control for protein load, the nitrocellulose membrane was Ponceau-stained before the immunological detection. From this staining, random, unspecified bands are shown as loading control (LC, lower panel).
(B) Immunofluorescence micrographs of eye3-2 mutant and its background strain 137c labeled with antibodies against SOUL3 (magenta) and acetylated-α-Tubulin (green) (upper panel) and labeled with antibodies against ChR1 (magenta) and acetylated-α-Tubulin (green) (lower panel). Scale bars = 5 µm.
Molecular Plant 2013 6, DOI: ( /mp/sss137)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

6. Figure 5 SOUL3 Knockdown Strains Show an Aberrant Eyespot Position.
(A) Crude extracts of wild-type and SOUL3 amiRNA lines (amiSOUL3) were prepared and different amounts of proteins from wild-type cells (100, 50, and 25 µg per lane) as well as 100 µg protein from crude extracts of amiSOUL3 lines were separated by 10% SDS–PAGE along with molecular mass standards and immunoblotted with anti-SOUL3 antibody. To control for protein load, the nitrocellulose membrane was Ponceau-stained before the immunological detection. From this staining, random, unspecified bands are shown as loading control (LC, lower panel). Quantification of SOUL3 protein levels were done as described in Figure 2B (n = 3) and are listed in Table 1.
(B) Bright field microscopy of different transgenic amiSOUL3C. reinhardtii lines along with wild-type and a vector control lacking the amiSOUL3 construct. The arrows indicate the position of the eyespot; an asterisk marks the pyrenoid. Scale bar = 5 µm.
(C) Immunofluorescence micrographs of transgenic amiSOUL3 lines with wild-type and vector control. Cells were stained with antibodies against ChR1 (magenta color) and acetylated-α-Tubulin (green color). The white arrows point to the stained D4 rootlet reaching to the eyespot. Scale bars = 5 µm.
Molecular Plant 2013 6, DOI: ( /mp/sss137)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
AmiSOUL3 Lines 56 and 151 Show a Different Phototactic Behavior than Wild-Type and Vector Control.C. reinhardtii wild-type and transgenic amiSOUL3 lines along with a vector control were distributed evenly to culture flasks (Nunc®) positioned horizontally, and exposed to a dim light gradient ranging from 11 µmol m–2 s–1 (left side) to not measurable low light intensities (right side). Flasks were left under these dim light conditions. Photographs of the different cultures were taken directly from above during the middle of the day and night phases. For the different time points, the same culture flask is shown for each strain.
Molecular Plant 2013 6, DOI: ( /mp/sss137)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions