Figure 1.

Transgene constructs and mutant K6 properties. (A) Structure of the lacZ transgenes. Both constructs feature the lacZ reporter sequence, modified to contain a nuclear localization signal (nls) at its N terminus, and a simian virus 40 poly(A) sequence at the 3′ end. They differ with respect to the amount of 5′upstream sequence from the hK6a gene. (B) Predicted secondary structure of hK6a as deduced from the cDNA sequence (7). A centrally located rod domain features four subdomains with heptad repeats of apolar residues and α-helical structure (1A, 1B, 2A, and 2B). The rod domain is flanked by non-helical head and tail domains. The internal deletion (Δ) encompasses the last 36 residues of the head domain and the first 16 residues of the rod domain. (C and D) Electron micrographs of negatively stained filaments assembled in vitro from purified recombinant keratins. (C) Filaments assembled from K6a-myc, K17, K5, and K14 (≈1:1:1:1 ratio). (D) Mutant filament assembled from ΔK6a-myc, K17, K5, and K14. Assemblies were subjected to a pelleting assay (9) to assess polymerization efficiency. The control keratin sample polymerized with >80% efficiency, while the mutant ΔK6a-myc containing sample polymerized with 40% efficiency. (Bar = 200 nm.)