Major epiplasmic proteins of ciliates are articulins: cloning, recombinant expression, and structural characterization.

The cytoskeleton of certain protists comprises an extensive membrane skeleton, the epiplasm, which contributes to the cell shape and patterning of the species-specific cortical architecture. The isolated epiplasm of the ciliated protist Pseudomicrothorax dubius consists of two major groups of proteins with molecular masses of 78-80 kD and 11-13 kD, respectively. To characterize the structure of these proteins, peptide sequences of two major polypeptides (78-80 kD) as well as a cDNA representing the entire coding sequence of a minor and hitherto unidentified component (60 kD; p60) of the epiplasm have been determined. All three polypeptides share sequence similarities. They contain repeated valine- and proline-rich motifs of 12 residues with the consensus VPVP--V-V-V-. In p60 the central core domain consists of 24 tandemly repeated VPV motifs. Within the repeat motifs positively and negatively charged residues, when present, show an alternating pattern in register with the V and P positions. Recombinant p60 was purified in 8 M urea and dialyzed against buffer. Infrared spectroscopic measurements indicate 30% beta-sheet. Electron microscopy reveals short filamentous polymers with a rather homogenous diameter (approximately 15-20 nm), but variable lengths. The small polymers form thicker filaments, ribbons, and larger sheets or tubes. A core domain similar to that of P. dubius p60 is also found in the recently described epiplasmic proteins of the flagellate Euglena, the so-called articulins. Our results show that the members of this protein family are not restricted to flagellates, but are also present in the distantly related ciliates where they are major constituents of the epiplasm. Comparison of flagellate and ciliate articulins highlights common features of this novel family of cytoskeletal proteins.

Articulins and epiplasmins: two distinct classes of cytoskeletal proteins of the membrane skeleton in protists.

The cortex of ciliates, dinoflagellates and euglenoids comprises a unique structure called the epiplasm, implicated in pattern-forming processes of the cell cortex and in maintaining cell shape. Despite significant variation in the structural organization of their epiplasm and cortex, a novel type of cytoskeletal protein named articulin is the principal constituent of the epiplasm in the euglenoid Euglena and the ciliate Pseudomicrothorax. For another ciliate, Paramecium, epiplasmins, a group of polypeptides with common biochemical properties, are the major constituents of the epiplasm. Using molecular tools and affinity purification we have selected polyclonal antibodies and identified epitopes of monoclonal antibodies that identify epitopes characteristic of articulins and epiplasmins. With these antibodies we have analysed the occurrence of the two types of cytoskeletal proteins in a dinoflagellate, a euglenoid and several ciliates. Our results indicate that both articulins and epiplasmins are present in these organisms, suggesting that both contribute to the organization of the membrane skeleton in protists. Articulins and epiplasmins represent two distinct classes of cytoskeletal proteins, since different polypeptides were labeled by articulin core domain-specific or epiplasmin epitope-specific antibodies in each organism studied. In one case, a polypeptide in Pseudomicrothorax was identified that reacts with both articulin core domain-specific and with anti-epiplasmin monoclonal antibodies; however, the epiplasmin monoclonal antibody epitope was mapped to the C terminus of the polypeptide, well outside the central VPV-repeat core domain that contains the articulin monoclonal antibody epitope and that is the hallmark of the articulins.

Characterisation of two articulins, the major epiplasmic proteins comprising the membrane skeleton of the ciliate Pseudomicrothorax.

Most protists possess a unique membrane skeleton, the epiplasm, which is involved in pattern forming processes of the cell cortex and functions in maintaining cell shape. Articulins, a novel class of cytoskeletal proteins, are major constituents of the epiplasm. We have isolated cDNAs encoding the two major articulins of the ciliate Pseudomicrothorax dubius. Peptide sequence data confirm the identity of the cloned cDNAs encoding articulins 1 and 4. With the data presented here sequence information for all major articulins of ciliates as well as the distantly related euglenoids is available. Sequence comparison of the two newly characterised ciliate articulins with the previously determined sequences of p60, a minor articulin of the same species, and the two euglenoid articulins reveals general sequence principles and uncovers new features of this protein family. The hallmark of articulins is a central core domain of repetitive motifs of alternating valine and proline residues, the VPV-motif. These VPV-motif repeats are either 12-residues, or in some places, six residues long. Positively and negatively charged residues segregate in register with valine and proline positions. The VPV-motif is unique to articulins. The terminal domains flanking the core are generally hydrophobic and contain a series of hexa- or heptapeptide repeats rich in glycine and hydrophobic residues. The sequences of these short repeats are very similar in articulins of the same species but are not conserved between euglenoids and ciliates.