Linkage of human keratin genes

Two families of keratins, type I and type II, can be distinguished within the intermediate filament family of proteins, and at least 20 genes in the human genome code for the 20 known keratin proteins. In epithelial intermediate filaments, keratins from both families appear to be coordinately expressed. We have screened a library of human genomic DNA and have identified several cases of linkage among homologous and heterologous pairs of keratin genes. Genes coding for type I keratins were found linked to those coding for type II keratins. Linkage was discovered also among homologous genes coding for type I keratins and among genes encoding type II keratins. In addition, we found genes coding for glycine-rich keratins linked to genes coding for those that do not contain glycine-rich regions. Our results raise the possibility that all keratin genes are linked in a single region of the human genome.     

Keratin incorporation into intermediate filament networks is a rapid process

The properties of keratin-containing intermediate filament (IF) networks in vivo were studied following the microinjection of biotinylated keratin. Keratin-IFs were biotinylated, disassembled, and separated into type I and type II proteins by ion exchange chromatography. Recombination of these derivatized type I and type II keratins resulted in the formation of 10-nm diameter IF. The type I keratins were microinjected into epithelial cells and observed by immunofluorescence microscopy. Biotin-rich spots were found throughout the cytoplasm at 15-20 min after injection. Short biotinylated fibrous structures were seen at 30-45 min after injection, most of which colocalized with the endogenous bundles of IF (tono-filaments). By 1 1/2 to 2 h after microinjection, extensive biotinylated keratin IF-like networks were evident. These were highly coincident with the endogenous tonofilaments throughout the cell, including those at desmosomal junctions. These results suggest the existence of a relatively rapid subunit incorporation mechanism using numerous sites along the length of the endogenous tonofilament bundles. These observations support the idea that keratin-IFs are dynamic cytoskeletal elements.     

The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments

The four major keratins of normal human epidermis (molecular mass 50, 56.5, 58, and 65-67 kD) can be subdivided on the basis of charge into two subfamilies (acidic 50-kD and 56.5-kD keratins vs. relatively basic 58-kD and 65-67-kD keratins) or subdivided on the basis of co-expression into two "pairs" (50-kD/58-kD keratin pair synthesized by basal cells vs. 56.5-kD/65-67-kD keratin pair expressed in suprabasal cells). Acidic and basic subfamilies were separated by ion exchange chromatography in 8.5 M urea and tested for their ability to reassemble into 10-nm filaments in vitro. The two keratins in either subfamily did not reassemble into 10-nm filaments unless combined with members of the other subfamily. While electron microscopy of acidic and basic keratins equilibrated in 4.5 M urea showed that keratins within each subfamily formed distinct oligomeric structures, possibly representing precursors in filament assembly, chemical cross-linking followed by gel analysis revealed dimers and larger oligomers only when subfamilies were combined. In addition, among the four major keratins, the acidic 50-kD and basic 58-kD keratins showed preferential association even in 8.5 M urea, enabling us to isolate a 50-kD/58-kD keratin complex by gel filtration. This isolated 50-kD/58-kD keratin pair readily formed 10-nm filaments in vitro. These results demonstrate that in tissues containing multiple keratins, two keratins are sufficient for filament assembly, but one keratin from each subfamily is required. More importantly, these data provide the first evidence for the structural significance of specific co-expressed acidic/basic keratin pairs in the formation of epithelial 10-nm filaments.     

Molecular cloning and characterization of the Endo B cytokeratin expressed in preimplantation mouse embryos

A cDNA clone of a keratin-related, intermediate filament protein, designated Endo B, was constructed from size-fractionated parietal endodermal mRNA and characterized. The 1466-nucleotide cDNA insert contains an open reading frame of 1272 nucleotides that would result in 5' and 3' noncoding sequences of 54 and 60 nucleotides, respectively. The predicted amino acid composition, molecular weight (47,400), and peptide pattern correlate well with data obtained on the isolated protein. The predicted amino acid sequence fits easily into the general domain structure suggested for all intermediate filament proteins with a unique amino-terminal head domain, a large conserved central domain of predominantly alpha-helical structure, and a relatively unique carboxyl-terminal or tail domain. Over the entire molecule, Endo B is 43% identical with human 52-kDa epidermal type I keratin. However, over two of the three regions contained in the central domain that are predicted to form coiled-coil structures, the Endo B is 54-68% identical with other type I keratin sequences. This homology, along with the presence of the completely conserved sequence DNARLAADDFR-KYE, which is found in all type I keratins, permits the unambiguous identification of Endo B as a type I keratin. Comparison of the Endo B sequence to other intermediate filament proteins reveals 22 residues which are identical in all intermediate filament proteins regardless of whether filament formation requires only one type of protein subunit (vimentin, desmin, glial fibrillar acidic protein, or a neurofilament protein) or two dissimilar types (type I and type II keratins). Endo B mRNA was detectable in RNA isolated from F9 cells treated with retinoic acid for 48 h. Approximately three to five genes homologous to Endo B were detected in the mouse genome.     

Terrestrial vertebrates have two keratin gene clusters; striking differences in teleost fish

Keratins I and II form the largest subgroups of mammalian intermediate filament (IF) proteins and account as obligatory heteropolymers for the keratin filaments of epithelia. All human type I genes except for the K18 gene are clustered on chromosome 17q21, while all type II genes form a cluster on chromosome 12q13, that ends with the type I gene K18. Highly related keratin gene clusters are found in rat and mouse. Since fish seem to lack a keratin II cluster we screened the recently established draft genomes of a bird (chicken) and an amphibian (Xenopus). The results show that keratin I and II gene clusters are a feature of all terrestrial vertebrates. Because hair with its multiple hair keratins and inner root sheath keratins is a mammalian acquisition, the keratin gene clusters of chicken and Xenopus tropicalis have only about half the number of genes found in mammals. Within the type I clusters all genes have the same orientation. In type II clusters there is a rare gene of opposite orientation. Finally we show that the genes for keratins 8 and 18, which are the first expression pair in embryology, are not only adjacent in mammals, but also in Xenopus and three different fish. Thus neighboring K8 and K18 genes seem a feature shared by all vertebrates. In contrast to the two well defined keratin gene clusters of terrestrial vertebrates, three teleost fish show an excess of type I over type II genes, the lack of a keratin type II gene cluster and a striking dispersal of type I genes, that are probably the result of the teleost-specific whole genome duplication followed by a massive gene loss. This raises the question whether keratin gene clusters extend beyond the ancestral bony vertebrate to cartilage fish and lamprey. We also analyzed the complement of non-keratin IF genes of the chicken. Surprisingly, an additional nuclear lamin gene, previously overlooked by cDNA cloning, is documented on chromosome 10. The two splice variants closely resemble the lamin LIII a + b of amphibia and fish. This lamin gene is lost on the mammalian lineage.     

Retrovirus-mediated transgenic keratin expression in cultured fibroblasts: specific domain functions in keratin stabilization and filament formation.

With retrovirus-mediated gene transfer, we used intact and deleted keratin proteins to investigate the molecular basis of intermediate filament function. Three levels of assembly show a different stringency for the involvement of individual keratin domains: protein accumulation requires the alpha helix domains; stable filament formation additionally requires both N- and C-terminal domains of either one of the two interacting keratins, suggesting that head to tail homotypic interaction is important for effective elongation; and higher order organization of the cytoplasmic network depends on correct type I-type II pairing of keratins. The presence of two distinct interaction sites along potentially different axes may explain the characteristic morphology of keratin intermediate filament networks.     

Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins

In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution- binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.     

'Hard' and 'soft' principles defining the structure, function and regulation of keratin intermediate filaments.

Keratins make up the largest subgroup of intermediate filament proteins and represent the most abundant proteins in epithelial cells. They exist as highly dynamic networks of cytoplasmic 10-12 nm filaments that are obligate heteropolymers involving type I and type II keratins. The primary function of keratins is to protect epithelial cells from mechanical and nonmechanical stresses that result in cell death. Other emerging functions include roles in cell signaling, the stress response and apoptosis, as well as unique roles that are keratin specific and tissue specific. The role of keratins in a number of human skin, hair, ocular, oral and liver diseases is now established and meshes well with the evidence gathered from transgenic mouse models. The phenotypes associated with defects in keratin proteins are subject to significant modulation by functional redundancy within the family and modifier genes as well. Keratin filaments undergo complex regulation involving post-translational modifications and interactions with self and with various classes of associated proteins.     

Localization of the gene for human simple epithelial keratin 18 to chromosome 12 using polymerase chain reaction.

Many human genes encoding keratin intermediate filament proteins are clustered on chromosomes 17 (the type I genes) and 12 (the type II genes). Some have not yet been localized, notably the genes for the primary embryonic keratins 8 and 18, normally expressed in simple epithelia: this is because the numerous pseudogenes for these keratins have made it difficult to identify the true functional gene in each case. Through the use of human-specific primers from within introns of the published gene sequence for human type I keratin 18, human genomic DNA has been specifically amplified using the polymerase chain reaction. A single reaction product was obtained. DNA from a characterized series of mouse-human somatic cell hybrid lines was tested for the presence of sequences able to initiate the chain reaction from these primers, and the presence or absence of this genomic DNA PCR product allowed us to assign a gene for human keratin 18 to chromosome 12 unambiguously. This differs from the location of other human type I keratins on chromosome 17 and may indicate the early divergence of the genes for stratifying cell keratins from that of simple, or embryonic, keratin 18.     

Phosphorylation of keratin intermediate filaments by protein kinase C, by calmodulin-dependent protein kinase and by cAMP-dependent protein kinase.

Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type II keratin 8 (molecular mass 55 kDa; basic type) in a 1:1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by cAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2+/calmodulin-dependent protein kinase II were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.     

The epidermal intermediate filament proteins of tunicates are distant keratins; a polymerisation-competent hetero coiled coil of the Styela D protein and Xenopus keratin 8

Two novel cytoplasmic intermediate filament (IF) proteins (C and D) from the tunicate (urochordate) Styela are characterised as putative keratin orthologs. The coexpression of C and D in all epidermal cells and the obligatory heteropolymeric IF assembly of the recombinant proteins argue for keratin orthologs, but the sequences do not directly reveal which protein behaves as a keratin I or II ortholog. This problem is solved by the finding that keratin 8, a type II keratin from man or Xenopus, forms chimeric IF when mixed with Styela D. Mutant proteins of Styela D and keratin 8 with a single cysteine in equivalent positions show that these chimeric IF are, like vertebrate keratin filaments, based on the hetero coiled coil. We propose that Styela D retains, in spite of its strong sequence drift, important molecular features of type I keratins. By inference Styela C reflects a type II ortholog. We discuss that type I to III IF proteins are expressed along the chordate branch of metazoa.     

Characterisation and tissue-specific expression of the two keratin subfamilies of intermediate filament proteins in the cephalochordate Branchiostoma

The cloning of three intermediate filament proteins expressed at the gastrula stage (kl, Y1, X1) extends the size of the IF multigene family of Branchiostoma to at least 13 members. This is one of the largest protein families established for the lancelet. Sequence comparisons indicate five keratin orthologs, three of type I (E1, k1, Y1) and two of type II (E2, D1). This assignment is confirmed by the obligatory heteropolymeric polymerisation behaviour of the recombinant proteins. In line with the hetero-coiled-coil principle IF are formed by any stoichiometric mixture of type I and II keratin orthologs. In spite of the strong sequence drift chimeric IF are formed between K8, a human keratin II, and two of the lancelet type I keratins. We discuss whether the remaining 8 IF proteins reflect three additional and potentially cephalochordate-specific subfamilies. The tissue-specific expression patterns of the 5 keratins and some other IF proteins were analysed by immunofluorescence in the adult. Keratins are primarily present in ectodermally derived tissues. Developmental control of the expression of some IF proteins is observed, but three keratins (k1, Y1, D1) and an additional IF protein (X1) detected at the gastrula stage are expressed throughout the life cycle.     

A family of type I keratin genes and the homeobox-2 gene complex are closely linked to the rex locus on mouse chromosome 11

Type I and type II keratins are major constituents of intermediate filaments that play a fundamental role in the cytoskeletal network. By using both somatic cell hybrids and conventional and interspecific linkage crosses, several genes encoding type I keratins, including the epidermal keratin K10, were shown to be closely linked to the homeobox-2 complex and the rex locus on mouse chromosome 11. The absence of crossovers between type I keratin-encoding genes and rex (N = 239), a locus affecting hair development, raises the possibility that mutations at rex and neighboring loci affecting skin and hair development involve type I keratin genes.     

Elucidating the early stages of keratin filament assembly

Because of extraordinarily tight coiled-coil associations of type I and type II keratins, the composition and structure of keratin subunits has been difficult to determine. We report here the use of novel genetic and biochemical methods to explore the early stages of keratin filament assembly. Using bacterially expressed humans K5 and K14, we show that remarkably, these keratins behave as 1:1 complexes even in 9 M urea and in the presence of a reducing agent. Gel filtration chromatography and chemical cross-linking were used to identify heterodimers and heterotetramers as the most stable building blocks of keratin filament assembly. EM suggested that the dimer consists of a coiled-coil of K5 and K14 aligned in register and in parallel fashion, and the tetramer consists of two dimers in antiparallel fashion, without polarity. In 4 M urea, both end-to-end and lateral packing of tetramers occurred, leading to a variety of larger heteromeric complexes. The coexistence of multiple, higher-ordered associations under strongly denaturing conditions suggests that there may not be a serial sequence of events leading to the assembly of keratin intermediate filaments, but rather a number of associations may take place in parallel.     

Type II keratins are phosphorylated on a unique motif during stress and mitosis in tissues and cultured cells

Epithelial cell keratins make up the type I (K9-K20) and type II (K1-K8) intermediate filament proteins. In glandular epithelia, K8 becomes phosphorylated on S73 ((71)LLpSPL) in human cultured cells and tissues during stress, apoptosis, and mitosis. Of all known proteins, the context of the K8 S73 motif (LLS/TPL) is unique to type II keratins and is conserved in epidermal K5/K6, esophageal K4, and type II hair keratins, except that serine is replaced by threonine. Because knowledge regarding epidermal and esophageal keratin regulation is limited, we tested whether K4-K6 are phosphorylated on the LLTPL motif. K5 and K6 become phosphorylated in vitro on threonine by the stress-activated kinase p38. Site-specific anti-phosphokeratin antibodies to LLpTPL were generated, which demonstrated negligible basal K4-K6 phosphorylation. In contrast, treatment of primary keratinocytes and other cultured cells, and ex vivo skin and esophagus cultures, with serine/threonine phosphatase inhibitors causes a dramatic increase in K4-K6 LLpTPL phosphorylation. This phosphorylation is accompanied by keratin solubilization, filament reorganization, and collapse. K5/K6 LLTPL phosphorylation occurs in vivo during mitosis and apoptosis induced by UV light or anisomycin, and in human psoriatic skin and squamous cell carcinoma. In conclusion, type II keratins of proliferating epithelia undergo phosphorylation at a unique and conserved motif as part of physiological mitotic and stress-related signals.     

Amino acid sequences of mouse and human epidermal type II keratins of Mr 67,000 provide a systematic basis for the structural and functional diversity of the end domains of keratin intermediate filament subunits

From the nucleotide sequences of specific cDNA clones, we present partial amino acid sequences (75-90% of the total) of 67-kDa type II keratin subunits expressed in terminally differentiating mouse and human epidermis. Analysis of the sequence information reveals that their secondary structures conform to the pattern common for all intermediate filament (IF) subunits. Together with the previously published sequence of the mouse 59-kDa type I keratin (Steinert, P. M., Rice, R. H., Roop, D. R., Trus, B. L., and Steven, A. C. (1983) Nature 302, 794-800) these data allow us to make comparisons between two keratins which are coexpressed in an epithelial cell type and which coassemble into the same IF. Moreover, these comparisons suggest a systematic plan for the general organization of the end domains of other keratin subunits. We postulate that each end domain consists of a set of subdomains which are distributed with bilateral symmetry with respect to the central alpha-helical domain. Type II (but not type I) keratins contain short globular sequences, H1 and H2, immediately adjacent to the central domain, that have been conserved in size and sequence and which account for most of the difference in mass between coexpressed type II and type I keratins. These are flanked by subdomains V1 and V2 that are highly variable in both length and sequence, often contain tandem peptide repeats, and are conspicuously rich in glycines and/or serines. At the termini are strongly basic subdomains (N and C, respectively) that are variable in sequence. Among keratins of a given type, their variability in mass appears to reside in the size of their V1 and V2 subdomains. However, coexpressed type I and type II keratins have generally similar V1 and/or V2 sequences. By virtue of the ease with which large portions of these subdomain sequences can be removed from intact keratin IF by limited proteolysis, we hypothesize that they lie on the periphery of the IF where they participate in interactions with other constituents of epithelial cells.     

Characterization of the major physiologic phosphorylation site of human keratin 19 and its role in filament organization

Keratin polypeptide 19 (K19) is a type I intermediate filament protein that is expressed in stratified and simple-type epithelia. Little is known regarding K19 regulation or function, and the only other type I keratin that has been studied in terms of regulation is keratin 18 (K18). We characterized K19 phosphorylation as a handle to study its function. In vivo, serine is the major phosphorylated residue, and phosphopeptide mapping of 32PO4-labeled K19 generates one major phosphopeptide. Edman degradation suggested that the radiolabeled phosphopeptide represents K19 Ser-10 and/or Ser-35 phosphorylation. Mutation of Ser-10 or Ser-35 followed by transfection confirmed that Ser-35 is the major K19 phosphorylation site. Transfection of Ser-35 --> Ala K19 showed a filament assembly defect as compared with normal or with Ser-10 --> Ala K19. Comparison of K18 and K19 phosphorylation features in interphase cells showed that both are phosphorylated primarily at a single site, preferentially in the soluble versus the insoluble keratin fractions. K19 has higher basal phosphorylation, whereas K18 phosphorylation is far more sensitive to phosphatase type I and IIA inhibition. Our results demonstrate that Ser-35 is the major K19 interphase phosphorylation site and that it plays a role in keratin filament assembly. K19 and K18 phosphorylations share some features but also have distinct properties that suggest different regulation of type I keratins within the same cells.     

Genes coding for intermediate filament proteins closely related to the hagfish "thread keratins (TK)" alpha and gamma also exist in lamprey, teleosts and amphibians

The "thread keratins (TK)" alpha and gamma so far have been considered highly specialized intermediate filament (IF) proteins restricted to hagfish. From lamprey, we now have sequenced five novel IF proteins closely related to TKalpha and TKgamma, respectively. Moreover, we have detected corresponding sequences in EST and genomic databases of teleosts and amphibians. The structure of the TKalpha genes and the positions of their deduced amino acid sequences in a phylogenetic tree clearly support their classification as type II keratins. The genes encoding TKgamma show a structure typical for type III IF proteins, whereas their positions in phylogenetic trees favor a close relationship to the type I keratins. Considering that most keratin-like sequences detected in the lancelet also exhibit a gene structure typical for type III IF proteins, it seems likely that the keratin gene(s) originated from an ancient type III IF protein gene. According to EST analyses, the expression of the thread keratins in teleost fish and amphibians may be particularly restricted to larval stages, which, in conjunction with the observed absence of TKalpha and TKgamma genes in any of the available Amniota databases, indicates a thread keratin function closely related to larval development in an aquatic environment.     

Cytoskeletal disorders in human keratinocytes--epidermolysis bullosa simplex]

The cytoskeletons possibly related to pathogenesis in skin disease may be limited to keratin intermediate filaments (KIF) in epidermal keratinocytes. Keratins are divided into two subclasses; 11 acidic (type I) keratins and 8 basic (type II) keratins. Combination of equimolar amounts of type I and type II can form KIF. KIFs in human epidermal basal cells consist of a pair of type I and type II keratins specifically synthesized in the basal cells, and those in spinous cells contain two pairs of keratin; a pair of basal cell keratin and another pair of keratin specific for suprabasal cells. In the first section, molecular biology and differentiation of keratins are reviewed. In the second section, epidermolysis bullosa simplex (EBS) was introduced from the view point of abnormal organization of KIFs. In the epidermis of EBS, clefts are induced in the basal cells by minor trauma or frictions consequently to produce bullae. Electron microscopy reveals small spherical aggregations of tonofilaments (KIFs) in the basal cells. In biopsies, these KIF aggregations might be caused by artifacts during procedures for biopsies, so that, in order to avoid these artifacts, we studied the KIF organization in cultured keratinocytes from a patient by immunofluorescence using anti-keratin antibodies and electron microscopy. Anti-keratin antibodies revealed a formation of small droplet-like aggregations of KIFs in many cultured cells adhering to the culture bottles, which were also suggested by electron microscopy. From these observations, it is suggested that the abnormal organization (droplets) of KIFs might be one of intrinsic factors for the pathogenesis of EBS.(ABSTRACT TRUNCATED AT 250 WORDS)     

The two-chain coiled-coil molecule of native epidermal keratin intermediate filaments is a type I-type II heterodimer.

The composition of the two-chain coiled-coil molecule of murine epidermal keratin intermediate filaments (KIF) containing keratins 1 (type II) and 10 (type I) has been explored using native-type KIF as well as KIF reassembled in vitro from protein dissolved in urea solutions or from mixtures of 3H-labeled and unlabeled purified chains. By use of cross-linking, high resolution polyacrylamide gel electrophoresis and blotting for 3H-labeled keratins or with an anti-mouse keratin 10 antibody, it was found that individual keratin chains form type I or type II homodimers and homotetramers in solution that do not assemble into KIF in vitro. When mixed in urea solutions of 5 M or greater, such homo-oligomers rapidly rearrange into mostly heterodimers and heterotetramers that support filament assembly. On the other hand, prekeratin, isolated from newborn mouse epidermis with 0.1 M sodium citrate buffer, pH 2.6, under conditions that do not dissociate the native coiled-coil molecule, consists exclusively of type I-II heterodimers and heterotetramers. It is necessary to dissolve prekeratin in 8-9.5 M urea for several hours in order to dissociate the native heterodimer molecule and incorporate tracer amounts of a single 3H-labeled keratin chain. These data establish that native KIF consist of heterodimer coiled-coil molecules. Furthermore, heterodimers are much more stable than homodimers and are the favored form of association in solution. However, some homodimers (10-30% of total) always form after dissolution in concentrated urea and can be assimilated into KIF during reassembly in vitro. The isolation of alpha-helix-enriched dimer particles from the 2B region of the rod domains upon limited proteolysis confirmed the presence of mostly heterodimer and some homodimer molecules in reassembled KIF. These properties of keratin chains in urea solutions hereby clarify a number of conflicting reports in the literature concerning the composition of the coiled-coil molecule. The presence of some homo-oligomeric species in reassembled KIF correlates with earlier observations of polymorphism as well as stoichiometry.