MULTIMER FORMATION AS A CONSEQUENCE OF SEPARATE HOMODIMERIZATION DOMAINS - THE HUMAN C-JUN LEUCINE-ZIPPER IS A TRANSPLANTABLE DIMERIZATION MODULE

Human c-Jun and c-Fos leucine zipper domains were examined for their a bility to serve as autonomous dimerization domains as part of a hetero logous protein construct. Schistosoma japonicum glutathione S-transfer ase (GST) was fused to recombinant Jun leucine zipper (rJunLZ) and Fos leucine zipper (rFosLZ) domains, SDS-PAGE 'snapshot' analyses based o n disulphide linkage of monomers demonstrated the ability of rJunLZ to function as a dimerization motif in a foreign protein environment, St eric hindrance prevented formation of rJunLZ-GST::rFosLZ-GST heterodim ers whereas rJunLZ-GST::rFosLZ and rJunLZ::rFosLZ-GST formed readily. Furthermore, rJunLZ-GST generated homodimers suggesting fusion protein heterodimers interact differently to homodimers, Gel filtration chrom atography confirmed that GST is a diner in solution and that attachmen t of a leucine zipper domain allows further interactions to take place , Sedimentation equilibrium analyses showed that GST is a stable dimer (K-a > 10(6) M(-1)) with no higher multimeric forms, rFosLZ-GST weakl y associates beyond a dimer (K-a similar to-4 x 10(5) M(-1)) and rJunL Z-GST associates indefinitely (K-a similar to 4 x 10(6) M(-1)), consis tent with an isodesmic model of association. The interaction of these leucine zippers independently of GST association demonstrates their ut ility in the modification of proteins when multimer formation is desir ed.