All IF Proteins Have a Conserved Core Domain and Are Organized Similarly into Filaments

Besides having in common an ability to form filaments 10 nm in diameter, all IF subunit proteins have a common do-

^ EXPERIMENTAL FIGURE 19-33 Electron microscopy visualizes intermediate structures in the assembly of intermediate filaments. Shown here are electron micrographs and drawings of IF protein dimers and tetramers and of mature intermediate filaments from Ascaris, an intestinal parasitic worm. (a) IF proteins form parallel dimers with a highly conserved coiled-coil core domain and globular tails and heads, which are variable in length and sequence. (b) A tetramer is formed by antiparallel, staggered side-by-side aggregation of two identical dimers. (c) Tetramers aggregate end-to-end and laterally into a protofibril. In a mature filament, consisting of four protofibrils, the globular domains form beaded clusters on the surface. [Adapted from N. Geisler et al., 1998, J. Mol. Biol. 282:601; courtesy of Ueli Aebi.]

main structure: a central a-helical core flanked by globular N- and C-terminal domains. The core helical domain, which is conserved among all IF proteins, consists of four long a helices separated by three nonhelical, "spacer" regions. The a-helical segments pair to form a coiled-coil dimer.

In electron micrographs, an IF-protein dimer appears as a rodlike molecule with globular domains at the ends; two dimers associate laterally into a tetramer (Figure 19-33a, b). The results of labeling experiments with antibodies to the N- or C-terminal domain indicate that the polypeptide chains are parallel in a dimer, whereas the dimers in a tetramer have an antiparallel orientation. The next steps in assembly are not well understood but seem to include the end-to-end association of tetramers to form long protofilaments, which aggregate laterally into a loose bundle of protofibrils. Compaction of a protofibril yields a mature 10-nm-diameter filament with the N- and C-terminal globular domains of the tetramers forming beaded clusters along the surface (Figure 19-33c). Interestingly, because the tetramer is symmetric, an intermediate filament may not have a polarity, as does an actin filament or a microtubule. This idea is supported by findings from experiments showing that vimentin subunits can incorporate along the length, as well as the ends, of a filament.

Although the a-helical core is common to all IF proteins, the N- and C-terminal domains of different types of IF proteins vary greatly in molecular weight and sequence. Partly because of this lack of sequence conservation, scientists initially speculated that the N- and C-terminal domains do not have roles in IF assembly. The results of several subsequent experiments, however, proved this hypothesis to be partly incorrect. For instance, if the N-terminal domain of an IF

protein is shortened, either by proteolysis or by deletion mu-tagenesis, the truncated protein cannot assemble into filaments. (Keratins are an exception; they form filaments even if both terminal domains are absent.) The prevailing view now is that the N-terminal domain plays an important role in the assembly of most intermediate filaments. Even though the C-terminal domain is dispensable for IF assembly, it seems to affect the organization of IF cytoskeletons in a cell. Thus these domains may control lateral interactions within an intermediate filament, as well as interactions between intermediate filaments and other cellular components.

Identity of IF Subunits Whether IF monomers, dimers, or tetramers constitute the immediate subunit for assembly of filaments, analogous to G-actin monomers in the assembly of microfilaments, is still unresolved. The main supporting evidence for the involvement of the IF tetramer comes from cell fractionation experiments showing that, although most vimentin in cultured fibroblasts is polymerized into filaments, 1-5 percent of the protein exists as a soluble pool of tetramers. The presence of a tetramer pool suggests that vimentin monomers are rapidly converted into dimers, which rapidly form tetramers.

Homo- and Heteropolymeric Filaments Some IF proteins form homopolymeric filaments; others form only het-eropolymeric filaments with other proteins in their class; and some can form both homo- and heteropolymeric filaments. Some IF proteins, but not the keratins, can form heteropolymers with IF proteins in another class. NF-L self-associates to form a homopolymer, but NF-H and NF-M commonly co-assemble with the NF-L backbone, and so most neurofilaments contain all three proteins. Spacer sequences in the coiled-coil regions of IF dimers or sequences in the diverse Nor C-terminal domains or both are most likely responsible for determining whether particular IF proteins assemble into heteropolymers or homopolymers. In fact, mutations in these regions generate mutated IF polypeptides that can form hetero-oligomers with normal IF proteins. These hybrid molecules often "poison" IF polymerization by blocking assembly at an intermediate stage. The ability of mutated IF proteins to block IF assembly has proved extremely useful in studies of the function of intermediate filaments in a cell. At the end of the chapter, we look at how such mutations in keratins have revealed the role of keratin filaments in the epidermis.

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