Protein-protein interactions between large proteins: two-hybrid screening using a functionally classified library composed of long cDNAs.
Large proteins have multiple domains that are potentially capable of binding many kinds of partners. It is conceivable, therefore, that such proteins could function as an intricate framework of assembly protein complexes. To comprehensively study protein-protein interactions between large KIAA proteins, we have constructed a library composed of 1087 KIAA cDNA clones based on prior functional classifications done in silico. We were guided by two principles that raise the success rate for detecting interactions per tested combination: we avoided testing low-probability combinations, and reduced the number of potential false negatives that arise from the fact that large proteins cannot reliably be expressed in yeast. The latter was addressed by constructing a cDNA library comprised of random fragments encoding large proteins. Cytoplasmic domains of KIAA transmembrane proteins (>1000 amino acids) were used as bait for yeast two-hybrid screening. Our analyses reveal that several KIAA proteins bearing a transmembrane region have the capability of binding to other KIAA proteins containing domains (e.g., PDZ, SH3, rhoGEF, and spectrin) known to be localized to highly specialized submembranous sites, indicating that they participate in cellular junction formation, receptor or channel clustering, and intracellular signaling events. Our representative library should be a very useful resource for detecting previously unidentified interactions because it complements conventional expression libraries, which seldom contain large cDNAs.
Nakayama
Manabu
M
Department of Human Gene Research, Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan. nmanabu@kazusa.or.jp
Kikuno
Reiko
R
Ohara
Osamu
O
D001923:Brain Chemistry / Q000235:genetics; D003001:Cloning, Molecular; D019295:Computational Biology / Q000295:instrumentation / Q000379:methods; D003593:Cytoplasm / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D007951:Leukemia, Myeloid / Q000235:genetics / Q000473:pathology; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000235:genetics / Q000502:physiology; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary / Q000502:physiology; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D012984:Software; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology
D018076:DNA, Complementary; D010455:Peptides; D011506:Proteins