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The Arabidopsis dynamin-like proteins ADL1C and ADL1E play a critical role in mitochondrial morphogenesis - PubMed

The Arabidopsis dynamin-like proteins ADL1C and ADL1E play a critical role in mitochondrial morphogenesis

Jing Bo Jin et al. Plant Cell. 2003 Oct.

Abstract

Dynamin-related proteins are high molecular weight GTP binding proteins and have been implicated in various biological processes. Here, we report the functional characterization of two dynamin homologs in Arabidopsis, Arabidopsis dynamin-like 1C (ADL1C) and Arabidopsis dynamin-like 1E (ADL1E). ADL1C and ADL1E show a high degree of amino acid sequence similarity with members of the dynamin family. However, both proteins lack the C-terminal Pro-rich domain and the pleckstrin homology domain. Expression of the dominant-negative mutant ADL1C[K48E] in protoplasts obtained from leaf cells caused abnormal mitochondrial elongation. Also, a T-DNA insertion mutation at the ADL1E gene caused abnormal mitochondrial elongation that was rescued by the transient expression of ADL1C and ADL1E in protoplasts. In immunohistochemistry and in vivo targeting experiments in Arabidopsis protoplasts, ADL1C and ADL1E appeared as numerous speckles and the two proteins colocalized. These speckles were partially colocalized with F1-ATPase-gamma:RFP, a mitochondrial marker, and ADL2b localized at the tip of mitochondria. These results suggest that ADL1C and ADL1E may play a critical role in mitochondrial fission in plant cells.

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Figures

Figure 1.
Figure 1.

Immunohistochemical Localization of ADL1E. (A) Expression of T7:ADL1E in transgenic plants. Total protein (10 μg) obtained from leaf tissues of nontransgenic wild-type (WT) or transgenic plants was separated on an SDS-polyacrylamide gel and transferred onto nylon polyvinylidene difluoride membranes. The blots were probed with a monoclonal anti-T7 antibody. (B) Localization of T7:ADL1E. Root protoplasts of nontransgenic wild-type (a) or transgenic (b) Arabidopsis grown in liquid medium for 1 week were prepared for immunohistochemistry. Root tip cells were labeled with the monoclonal anti-T7 antibody, and lissamine rhodamine–labeled anti-mouse IgG was used as the secondary antibody. Bar = 10 μm.

Figure 2.
Figure 2.

In Vivo Localization of ADL1C and ADL1E. (A) Patterns of GFP-fused ADL1C and ADL1E. Protoplasts were transformed with the constructs indicated, and the localization of these proteins was examined 24 to 48 h after transformation. Bars = 20 μm. (B) Colocalization of ADL1C and ADL1E. Protoplasts were transformed with the constructs indicated, and the localization of these fusion proteins was examined. Yellow signal indicates overlap between the green and red fluorescent signals. Bars = 20 μm. (C) Colocalization of T7-tagged ADL1E with GFP-tagged ADL1C or ADL1E. Protoplasts obtained from transgenic plants harboring T7:ADL1E were transformed with the constructs indicated, and the localization of these proteins was examined by immunohistochemistry. T7:ADL1E was detected from the fixed protoplasts using anti-T7 antibody, whereas GFP-fused proteins were observed directly by green fluorescence. Bars = 20 μm.

Figure 3.
Figure 3.

Elongation of Mitochondria in the Presence of Overexpressed ADL1C[K48E]. (A) Protoplasts were transformed with F1-ATPase-γ:RFP plus pCaMV (a), F1-ATPase-γ:RFP plus T7:ADL1C (b), or F1-ATPase-γ:RFP plus T7:ADL1C[K48E] (c), and the fluorescence emitted by RFP was detected 24 to 48 h after transformation. Bars = 20 μm. (B) Quantification of protoplasts based on mitochondrial morphology. Protoplasts were transformed with F1-ATPase-γ:RFP plus pCaMV (bars 1), F1-ATPase-γ:RFP plus T7:ADL1C (bars 2), F1-ATPase-γ:RFP plus T7:ADL1C[K48E] (bars 3), or F1-ATPase-γ:RFP plus HA:ADL1C plus T7:ADL1[K48E] (bars 4). The number of protoplasts was counted based on the mitochondrial morphology at 48 h after transformation. The punctate staining pattern (shown in [Aa] and [Ab]) was considered normal, whereas the elongated pattern (shown in [Ac]) was considered abnormal. More than 30 randomly selected protoplasts were counted at each time for each condition. The transformation experiment was performed three times with identical conditions. (C) Protein gel blot analysis. Protein extracts were obtained from protoplasts transformed with F1-ATPase-γ:RFP plus pCaMV (lane 1), F1-ATPase-γ:RFP plus T7:ADL1C (lane 2), F1-ATPase-γ:RFP plus T7:ADL1C[K48E] (lane 3), or F1-ATPase-γ:RFP plus HA:ADL1C plus T7:ADL1[K48E] (lane 4) and examined by protein gel blot analysis using the monoclonal anti-T7 (T7 mAb), anti-HA (HA mAb), and anti-RFP (RFP mAb) antibodies for T7-tagged ADL1C, HA-tagged ADL1C, and F1-ATPase-γ:RFP, respectively. pCaMV was included in the transformation as a control DNA.

Figure 4.
Figure 4.

Mitochondria Are Elongated in adl1e Mutants. Ultrathin sections prepared from leaf tissues of wild-type ([A] and [C]) and adl1e mutant ([B] and [D] to [F]) plants were examined by transmission electron microscopy. CH, chloroplast; CW, cell wall; IS, intercellular space; M, mitochondria. Bars = 2 μm for (A) and (B) and 200 nm for (C) to (F).

Figure 5.
Figure 5.

Abnormal Morphology of Mitochondria in adl1e Mutants Can Be Rescued by Transient Expression of ADL1C and ADL1E in Protoplasts. (A) Complementation of adl1e by transiently expressed ADL1E. Protoplasts were prepared from adl1e mutant plants and transformed with F-ATPase-γ:RFP alone or together with T7:ADL1E. As a control, protoplasts obtained from wild-type (WT) plants were transformed with F1-ATPase-γ:RFP. The fluorescence emitted by RFP was examined at various time points after transformation. Bars = 20 μm. (B) Quantification of morphological change. Protoplasts obtained from wild-type and adl1e mutant plants were transformed with F1-ATPase-γ:RFP (bars WT and 1), F1-ATPase-γ:RFP plus T7:ADL1E (bars 2), F1-ATPase-γ:RFP plus T7:ADL1C (bars 3), and F1-ATPase-γ:RFP plus T7:ADL1C[K48E] (bars 4). The number of protoplasts was counted based on the mitochondrial morphology indicated by RFP fluorescence. The mitochondria shown in (Aa) and (Ac) were considered to be in a punctate staining pattern, whereas the mitochondria shown in (Ab) were considered to be in an elongated pattern. More than 50 transformed cells were counted at each time point in triplicate experiments. The numbers and error bars indicate means and standard deviations, respectively. (C) Expression of T7:ADL1C and T7:ADL1E in protoplasts. Protein extracts were prepared from protoplasts transformed with F-ATPase-γ:RFP alone (lanes WT and 1) or together with T7:ADL1E (lane 2), ADL1C (lane 3), or ADL1C[K48E] (lane 4) and analyzed by protein gel blot analysis with anti-RFP (RFP mAb) and anti-T7 (T7 mAb) antibodies.

Figure 6.
Figure 6.

GFP:ADL1C Partially Colocalizes with F1-ATPase-γ:RFP and ADL2b. (A) Localization of ADL1C at mitochondria. Protoplasts were transformed with the indicated constructs, and the localization of these proteins was examined 24 to 48 h after transformation. Arrows indicate overlap of green and red fluorescent signals. Bars = 20 μm. (B) Partial colocalization of ADL1C with ADL2b in the mitochondria. Protoplasts were transformed with the indicated constructs, and the localization of the encoded proteins was examined by immunohistochemistry. HA-tagged proteins were detected with a monoclonal anti-HA antibody. GFP-tagged proteins were observed directly by green fluorescence. Arrows indicate the overlap between ADL2b:HA and GFP:ADL1C. Arrowheads indicate GFP:ADL1C-positive speckles that did not colocalize with ADL2b:HA. Bars = 20 μm. (C) Protein gel blot analysis of transiently expressed ADL isoforms. Protein extracts were prepared from protoplasts transformed with the indicated constructs and used for protein gel blot analysis using anti-HA antibody. WT, untransformed.

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