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doi: 10.1242/10.1242/jcs.00181

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Subcellular targeting of metabolic enzymes to titin in heart muscle may be mediated by DRAL/FHL-2

Stephan Lange^1,, Daniel Auerbach^1,*,, Patricia McLoughlin², Evelyne Perriard¹, Beat W. Schäfer², Jean-Claude Perriard^1, and Elisabeth Ehler¹

¹ Institute of Cell Biology, ETH Hönggerberg, 8093 Zurich, Switzerland
² Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zurich, 8032 Zurich, Switzerland
^* Present address: Dualsystems Biotech AG, Winterthurerstrasse 190, 8057 Zürich, Switzerland

View larger version (30K): [in a new window]   Fig. 5. Mapping of the DRAL/FHL-2-binding site in the N2B region of titin. Five different GFP-tagged deletion constructs of the N2B region were assayed for their interaction with DRAL/FHL-2 in pull-down assays. (A) Schematic representation of the employed constructs. (B) Investigation of the interaction between deletion constructs of the N2B region and DRAL by a GST pull-down assay. Only constructs that contain the residues between 3749 and 4019 of the unique sequence 3 are able to bind to DRAL/FHL-2-GST. The putative DRAL/FHL-2-binding site is indicated by a dotted line in the schematic drawing of the titin N2B region. (C) The putative DRAL/FHL-2-binding site alone shows proper targeting to the sarcomere, as shown in NRC double-transfected with GFP-N2B5 (a) and DRAL/FHL-2-FLAG (a'). Arrowheads in the insets point at the doublet flanking the Z-disc. (D) Coimmunoprecipitation of GFP-N2B5 and DRAL/FHL-2-FLAG. COS cells were cotransfected with GFP-N2B5 and DRAL/FHL-2-FLAG, immunprecipitation was carried out with a monoclonal anti-FLAG antibody followed by immunoblotting with a peroxidase-conjugated polyclonal rabbit anti-GFP antibody. The domain nomenclature and amino acid numbering correspond to that of the human cardiac titin sequence [X90568 (Labeit and Kolmerer, 1995)]. Bar, 10 µm.

View larger version (24K): [in a new window]   Fig. 1. Expression pattern of DRAL/FHL-2 in cardiac and skeletal muscle. (A) DRAL/FHL-2 is expressed only in cardiac muscle from adult mice. Immunoblots with a polyclonal rabbit anti-DRAL/FHL-2 antibody on whole muscle samples of adult mouse ventricle (lane 1), mouse tibialis anterior (fast twitch skeletal muscle, lane 2) and mouse soleus (slow twitch skeletal muscle, lane 3) show a band of the expected molecular weight of 34 kDa only in the heart. (B) DRAL/FHL-2 expression is upregulated in fetal mouse heart. Immunoblots with a polyclonal rabbit anti-DRAL/FHL-2 antibody on whole muscle samples of E12.5 (lane 1), E16.5 (lane 2), E18.5 (lane 3) and P0 (lane 4) heart show that DRAL/FHL-2 is expressed in cardiomyocytes during late embryonic development (upper panel). Equal amounts of heart tissue were loaded, as judged by the expression of -cardiac actin (bottom panel).

View larger version (83K): [in a new window]   Fig. 2. DRAL/FHL-2 colocalises with the N2B region of titin. (A) Schematic representation of antibody epitopes on titin. (B) Mapping of the exact position of DRAL/FHL-2 in the I-band by costaining with antibodies against different titin epitopes. Cardiac myofibrils from papillary muscle (a,b,c) or neonatal rat cardiomyocytes (d) were stained with an antibody directed against DRAL/FHL-2 (a', b', c', d'), together with antibodies directed against the N-terminus of titin (T12 epitope, a), the N2B region (I19 epitope, b), the N2A region (c) and against the PEVK region (9D10 epitope, d). DRAL/FHL-2 colocalises with the N2B region, but not with the other regions of titin. Arrowheads indicate the center of the Z-disc. Bar, 2 µm.

View larger version (127K): [in a new window]   Fig. 3. DRAL/FHL-2 interacts with the N2B region of titin in a yeast two-hybrid assay. Yeast strain L40 was cotransformed with LexA-DRAL/FHL-2 and Gal4AD-N2B (lane 1), LexA-lamin C and Gal4AD-N2B (lane 2), LexA and Gal4AD-N2B (lane 3) and LexA-DRAL/FHL-2 and Gal4AD (lane 4). Yeast transformants were streaked out on selective plates (a) and ß-galactosidase activity was detected using a filter assay (b). Results of two independent colonies per yeast transformation are shown. Only LexA-DRAL/FHL-2 in combination with Gal4AD-N2B yields any measurable ß-galactosidase activity (lane 1). N2B does not interact with an unrelated bait (LexA-lamin C, lane 2) or with LexA alone (lane 3) and LexA-DRAL/FHL-2 does not interact with Gal4AD alone (lane 4).

View larger version (49K): [in a new window]   Fig. 4. Confirmation of the interaction between DRAL/FHL-2 and the N2B and is2 regions of titin by colocalisation and pull-down experiments. (A) Transient transfection assays. GFP-N2B (a) and DRAL/FHL-2-FLAG (a') are localised in a similar pattern in transiently transfected NRC, with a broad doublet flanking the Z-disc (arrowheads) and a weaker striation at the M-band. A similar colocalisation can be observed in neonatal rat cardiomyocytes transiently transfected with GFP-is2 (b) and DRAL/FHL-2-FLAG (b'). (B) Pull-down assays: GFP-N2B and GFP-is2 interact with DRAL/FHL-2-GST but not with GST alone. Proteins in the supernatant and pellet fractions were detected by immunoblotting using a GFP antibody.

View larger version (44K): [in a new window]   Fig. 6. DRAL/FHL-2 interacts with the metabolic enzymes MM-CK, adenylate kinase and phosphofructokinase. (A) Transient transfection assays. GFP-tagged MM-CK (a), phosphofructokinase (b), and adenylate kinase (c) colocalise with DRAL/FHL-2-FLAG (a', b', c', d') in neonatal rat cardiomyocytes, with a strong signal around the Z-disc (arrowheads) and a weaker signal in the M-band (arrows). GFP-tagged Smpx/Csl (d) is also colocalised with DRAL/FHL-2. (B) Interaction of the metabolic enzymes with DRAL/FHL-2 in pull-down assays. MM-CK, adenlyate kinase and phosphofructokinase interact with DRAL/FHL-2-GST, but not with GST alone. Despite its similar localisation pattern in cardiomyocytes, Smpx/Csl does not interact with DRAL/FHL-2 in the pull-down assay. PFK, phosphofructokinase; AK1, adenylate kinase. Bar, 10 µm.

View larger version (21K): [in a new window]   Fig. 7. DRAL/FHL-2 interacts with the four and a half LIM domain protein FHL-1 but not with the LIM-only protein MLP. Pull-down assays: FLAG-tagged FHL-1 interacts with DRAL/FHL-2-GST but not with GST alone. In contrast, HA-tagged MLP shows no interaction with either GST-DRAL/FHL-2 or GST alone. The interaction of FHL-1 with DRAL/FHL-2 can be abolished by the addition of 10 mM EDTA, which unfolds LIM domains through the removal of the coordinated metal ions.

View larger version (8K): [in a new window]   Fig. 8. Schematic drawing of the N2B and is2 regions of titin and their associated proteins. DRAL/FHL-2 interacts directly with the N2B and is2 regions and serves as an adaptor molecule to couple the metabolic enzymes MM-CK, adenylate kinase and phosphofructokinase to titin. In addition, MM-CK may be bound to the M-band region of the sarcomere via its association with additional M-band proteins (not shown here). The domain nomenclature and amino acid numbering correspond to that of the human cardiac titin sequence [X90568 (Labeit and Kolmerer, 1995)].