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First published online 12 February 2003
doi: 10.1242/jcs.00309

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The lipoma preferred partner LPP interacts with -actinin

¹ ITI Research Institute, University of Bern, PO Box 54, CH-3010 Bern, Switzerland
² Institute for Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Versbacher Strasse 5, D-97078 Würzburg, Germany

View larger version (24K): [in a new window]   Fig. 1. Domain structures of the three proteins Trip6, zyxin and LPP. The N-terminal motif conserved in zyxin and LPP is indicated by a black bar in A. Note that this motif is not present in Trip6. The amino-acid sequences flanking the conserved motif are aligned in B. The sequences for mouse, human and chicken zyxin as well as human LPP are included. Identical residues are boxed.

View larger version (49K): [in a new window]   Fig. 2. Direct interaction of LPP and -actinin in a blot overlay. Full-length LPP (right) as well as its N-terminal sequence (residues 1-109, left) were expressed in bacteria as GST-fusion proteins and resolved on polyacrylamide gels (0.1-2 µg/lane). After transfer to nitrocellulose, the proteins were probed with radiolabeled -actinin. GST (2 µg/lane) and various protein standards were included as controls.

View larger version (18K): [in a new window]   Fig. 3. Mapping of the LPP interaction site in -actinin by the two-hybrid system. The yeast reporter strain Y190 was cotransfected with a prey vector encoding the N-terminus of LPP (residues 1-61) and different bait vectors encoding various fragments of -actinin as indicated. Two-hybrid interactions were analyzed by growth on histidine, tryptophan and leucine-deficient plates. Positive interactions (+) were verified by the colony filter lift assay. Interactions of the same -actinin fragments with the N-terminus of zyxin (residues 1-42) were analyzed in parallel experiments. Note that all -actinin fragments that revealed a positive interaction are able to form dimers in vitro (Li and Trueb, 2001). CH, region containing calponin homology domains; SPEC, region containing spectrin-like repeats; EF, region containing EF hands.

View larger version (22K): [in a new window]   Fig. 4. Competition between LPP and zyxin for the binding site of -actinin as revealed by the three-hybrid system. Yeast reporter strain Y190 was cotransfected with the prey vector pACT2 encoding the -actinin rod (residues 264-725) and the bait vector pBridge. The latter vector contained the sequence for zyxin (residues 1-42) in multiple cloning site I and the sequences for zyxin (residues 1-42) or LPP (residues 1-61 or 1-40) in multiple cloning site II. Note that multiple cloning site II is under the control of the MET25 promoter, which is active in the absence of methionine, but inactive in its presence. Colonies that grew on leucine- and tryptophan-deficient plates were inoculated into liquid medium lacking leucine and tryptophan (black bars) or, alternatively, lacking leucine, tryptophan and methionine (grey bars). Expression of the reporter gene for ß-galactosidase was analyzed by a colorimetric assay. The results represent the means with standard deviation from at least three independent determinations.

View larger version (110K): [in a new window]   Fig. 5. Comparison of the subcellular distribution of GFP-tagged LPP and zyxin. H9C2 cells were transiently transfected with a full-length cDNA for LPP ligated into the expression vector pEGFP. Two days after transfection, the cells were fixed, permeabilized and stained with antibodies against -actinin and zyxin, respectively. The green panel shows direct fluorescence emitted from GFP, the red panel indirect immunofluorescence from antibodies against -actinin or zyxin. Bar, 20 µm.

View larger version (101K): [in a new window]   Fig. 6. Recruitment of -actinin to LPP fusion proteins targeted to the surface of mitochondria. PtK2 cells were transiently transfected with GFP-fusion constructs encoding full-length LPP, full-length LPP lacking amino acids 41-57 (LPP), the N-terminal fragment of LPP (LPP61, residues 1-61) or a shorter fragment (LPP40, residues 1-40) as well as a mitochondrial anchor (M) as indicated. Two days after transfection, the cells were fixed, permeabilized and stained with antibodies against -actinin. The green panel shows direct fluorescence emitted from GFP, the red panel indirect immunofluorescence from antibodies against -actinin. Arrows point to large clusters of mitochondria. Bar, 20 µm.

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