1. Volume 19, Issue 2, Pages 151-156 (January 2009)
Plant-Specific Protein MCD1 Determines the Site of Chloroplast Division in Concert with Bacteria-Derived MinD 
Hiromitsu Nakanishi, Kenji Suzuki, Yukihiro Kabeya, Shin-ya Miyagishima 
Current Biology 
Volume 19, Issue 2, Pages (January 2009)
DOI: /j.cub
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2. Figure 1 Multiple Chloroplast Division in mcd1 Mutants
(A–D) Chloroplasts were observed by Nomarski optics in leaf mesophyll cells (A, B) and in petiole cells (C, D). Wild-type (A, C) and mcd1-1 mutant (B, D) are shown. Arrows indicate the constriction sites of dividing chloroplasts.
(E–H) Localization of FtsZ2-GFP (E, F) and GFP-DRP5B (G, H) in wild-type (E, G) and mcd1-1 mutant (F, H). The fluorescence of GFP is green and the autofluorescence of chlorophyll is red.
Each set of images (A and B, C and D, and E–H) are shown at the same magnification. Scale bars represent 5 μm.
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3. Figure 2
MCD1 Is an Integral Membrane Protein of the Chloroplast Inner Envelope
(A) Predicted primary structure of the MCD1 protein. The putative transit peptide, transmembrane domain, and coiled-coil domain are indicated with black boxes, and their positions within the amino acid sequences are indicated. aa indicates amino acids.
(B) Homogenate of the wild-type plants (Total) was centrifuged at 20,000 × g to sediment the low-speed pellet fraction (LSP). The supernatant fraction was centrifuged at 100,000 × g to separate the high-speed pellet (HSP) and supernatant fractions (S). 10 μg of protein were loaded in each lane.
(C) The low-speed pellet fraction was treated with 0.1 M sodium carbonate (Na2CO3) or 1% Nonidet P-40 (NP-40), and then separated at 100,000 × g into the pellet (P) and supernatant (S) fractions. 10 μg of protein were loaded in each lane.
(D) Isolated chloroplasts were lysed osmotically and separated into thylakoid and envelope fractions. The envelope membrane protein, TOC34, and thylakoid membrane protein, Lhcb1, were also detected as experimental controls. Isolated chloroplasts (2.5 μg proteins), the thylakoid fraction (0.5 μg proteins), and the envelope fraction (0.5 μg proteins) were loaded.
(E) Isolated chloroplasts were incubated in the absence or presence of thermolysin, trypsin, and 1% Triton X-100 (TX100). After quenching the reactions, the protein composition of the chloroplasts (containing 1 μg of chlorophyll) was analyzed by immunoblotting. The inner envelope protein, TIC40, the intermembrane space protein, TIC22, and the outer envelope protein, TOC34, were also examined as experimental controls.
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4. Figure 3
Localization of MCD1 at the Chloroplast Division Sites and in Punctate Structures on the Envelope Membrane
(A and B) Nomarski (A) and immunofluorescent (B) images of a section of a single wild-type leaf cell. The strong fluorescence signal indicates the localization of MCD1 detected by MCD1 antibodies.
(C–F) Localization of MCD1 in unconstricted (just before division; [C] and [E]) and constricted (during division; [D] and [F]) chloroplasts. Images were obtained by 1/2 s exposure ([C, D]; wild-type) or 1/10 of a second exposure ([E, F]; 35S-MCD1 transformant).
Images in (A) and (B) and in (C)–(F) are each shown at the same magnification. Scale bars represent 5 μm.
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5. Figure 4 MCD1-Dependent Localization of MinD
(A–E) Localization of MinD in wild-type, mcd1-1 mutant, and 35S-MCD1 transformant. The strong fluorescence signal indicates the localization of the MinD detected by MinD antibodies. A section of a single wild-type leaf cell was observed by immunofluorescence microscopy (A). Localization of MinD in unconstricted (just before division; [B]) and constricted (during division; [C]) chloroplasts of the wild-type. Localization of MinD in single chloroplasts of mcd1-1 (D) and of the 35S-MCD1 transformant (35S-MCD1; [E]). Images were obtained by 1/2 s exposure ([A–D]; wild-type and mcd1-1) or 1/10 of a second exposure ([E]; 35S-MCD1). Arrows indicate the localization of MinD at the division site. Images in (A) and (D) and in (B)–(C) and (E) are shown at the same magnification. Scale bar represents 5 μm.
(F) MinD transcript levels were examined by RT-PCR. ACTIN2 was used as the quantitative control.
(G) Levels of MinD were compared by immunoblotting. 50 μg of total protein extracted from young leaves of the wild-type, mcd1-1, and 35S-MCD1 transformant were analyzed with MinD antibodies.
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6. Figure 5
Effect of minD (arc11) Mutation and Overexpression on the Localization and Level of MCD1
(A) Proteins extracted from whole plants of the wild-type and minD, and then MinD level was analyzed by immunoblotting with MinD antibodies. 50 μg of protein were loaded in each lane.
(B) Localization of MinD in a single chloroplast of minD detected by MinD antibodies. Image was obtained by 1/2 s exposure. Scale bar represents 5 μm.
(C and D) Localization of MCD1 in a single chloroplast of minD (C) and in the MinD overexpresser (35S-MinD; [D]) was observed by immunofluorescence microscopy with MCD1 antibodies. Arrows indicate the ring structures. Sclae bar represents 5 μm. Image was obtained by 1/2 s exposure ([C]; minD) or 1/10 of a second exposure ([D]; 35S-MinD).
(E) Levels of MCD1 were compared by immunoblotting. 5 μg of total protein extracted from young leaves of the wild-type, minD, and MinD overexpresser (35S-MinD) were analyzed with MCD1 antibodies.
(F) MCD1 transcript levels were examined by RT-PCR. ACTIN2 was used as the quantitative control.
(G) Protein-protein interaction between MCD1 and MinD in the yeast two-hybrid assays. Yeast strain AH109 was transformed with paired constructs of protein fused to the GAL4 DNA binding domain (BD-MCD or BD-MinD63-326) and the GAL4 activation domain (AD, AD-MCD or AD-MinD63-326). Transformants were spotted at dilutions of 1:1, 1:10, 1:100, and 1:1000 (left to right) onto SD/-Leu/-Trp (-LT) or SD/-Leu/-Trp/-His (-LTH) plates.
Current Biology  , DOI: ( /j.cub )
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