Analysis of striated fiber formation by recombinant SF-assemblin in Vitro

Abstract

The basal bodies of green flagellates are often connected to striated microtubule-associated fibers (SMAFs), which are highly ordered bundles of 2 nm filaments. SF-assemblin (33 kDa) is the principal structural subunit of the SMAFs and consists of a non-helical head domain of approximately 32 residues and an α-helical rod domain that shows a pronounced coiled-coil forming ability. To investigate the functional role of the head domain we expressed N-terminally truncated molecules using a cDNA coding for SF-assemblin from Chlamydomonas reinhardtii. Recombinant wild-type SF-assemblin or molecules with an N-terminal deletion of ten residues formed striated fibers with an axial repeat of 28 nm. N-terminal truncations of 19 and 29 residues yielded assembly-incompetent molecules, revealing that the head domain is necessary for the constitution of striated fibers. Further, molecules with an internal deletion of 24 residues or with duplicated segments corresponding to insertions of 29 and 58 residues were constructed. The resulting fibers had altered cross-striation patterns and axial repeats. The observed shifts in the axial repeat corresponded well to the number of inserted or deleted residues, indicating a linear coherence between molecule length and axial repeat. The heptad pattern of the rod domain of SF-assemblin is regularly interrupted by skip residues. The removal of one or two skip residues had no significant effect on the ultrastructure of the striated fibers. Substitution of skip no. 2 with alanine resulted in a modified, asymmetric cross-striation pattern, indicating a polar architecture of the striated fibers. In summary, various mutations of SF-assemblin effected the solubility of the molecules, and the axial repeat, cross-striation pattern, or overall appearance of the fibers. Thus, analysis of SF-assemblin may represent a valuable system to study the interactions involved in the polymerization of fibrous coiled-coil proteins. A model of the SMAFs based on staggered protofilaments consisting of overlapping 36 nm subunits is presented.