Biochemical and Biophysical Research Communications
MAP, a protein interacting with a tumor suppressor, merlin, through the run domain☆
Section snippets
Materials and methods
Plasmids. The wild type (WT) merlin and its deletion mutants (M1–M4) were previously described in the mammalian system [16]. The yeast plasmids for the merlin proteins were generated by cloning the PCR fragments in pGBT9, a GAL4 DNA binding domain vector (Clontech, Palo Alto, CA) using the BamHI (both 5′ and 3′ primers) site. The full-length and derivatives of human MAP were cloned in the EcoRI/XhoI sites of pACT2, a GAL4 activation domain vector (Clontech, Palo Alto, CA) using PCR. The
Structural characteristics of MAP
To find the binding partners, the full-length merlin was used as bait for the yeast two-hybrid screening. Among the total of 2 × 106 transformants from a human brain cDNA library, 25 strong positive clones were finally selected. Partial nucleotide sequencing and homology searches (BLAST) revealed that five of the clones contained the same part of a hypothetical protein (BC008078) that we named MAP. A full sequence of MAP was obtained from the 5′-RACE screening of human placenta 5′ stretch cDNA.
Discussion
In this paper, using the full-length merlin as bait in the yeast two-hybrid screen, we identified a MAP. MAP is a protein of about 84.5 kDa which is expressed ubiquitously in many tissues and various cell lines (Fig. 2). The GST pull-down and immunoprecipitation experiments demonstrated the physical interaction of merlin and MAP in vitro and in vivo (Fig. 4, Fig. 5). Domain analyses identified the MAP RUN domain and the C-terminal region of merlin as responsible for their interaction (Fig. 3,
Acknowledgments
The authors thank other members of our laboratory for their valuable assistance. This study was supported by grant of the Korean Health 21 R&D Project, Ministry of Health Welfare, Republic of Korea (00-PJ3-PG6-GN02-0002, 02-PJ10-PG6-GP01-0002).
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How do you RUN on?
2011, FEBS LettersCitation Excerpt :The RUN domain containing proteins have been shown to promote endosomal fusion and are important for vesicular transport, and the RUN domains appear to be required for localization to detergent-insoluble endosomal microdomains [36–38]. The physical interaction between RUN proteins and filamentous materials has been confirmed by pull-down and two-hybrid experiments using wild type and mutant proteins [39–41]. However, it is noteworthy that interaction with Rab (or Rap) is not the sole structural feature linking RUN proteins to vesicle traffic.
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