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

Abstract

Abstract. 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% B-sheet. Electron microscopy reveals short filamentous polymers with a rather homogenous diameter (~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.T HE membrane skeleton of eukaryotic cells is part of the cytoskeleton. It is involved in the organization and maintenance of cell shape and functions in the generation and stabilization of specialized membrane domains. This submembraneous organization is usually dictated by micro filamentous structures containing actin and a variety of actin-binding proteins. Particularly well-analyzed examples are, for instance, the membrane skeletons of vertebrate red blood cells (for review see Bennett, 1990) and the intestinal brush border cells of various vertebrates (for review see Louvard, 1989). An actin-based subplasma membrane organization occurs not only in metazoan cells, but is also typical for some protists such as Dictyostelium and Physarum (for reviews see Schleicher and Noegel, 1992;Stockem and Brix, 1994).In contrast, other protists possess a unique cortical cytoplasm: the epiplasm. The epiplasm is always in close conAddress all correspondence to Irm Huttenlauch,

Plasmodium alveolins possess distinct but structurally and functionally related multi-repeat domains

“…3b ), albeit typically with lower probability scores. Articulins and plateins, cortical cytoskeleton proteins from Euglena and Euplotes spp., respectively, have also been reported to have 12-amino acid repetitive motifs rich in valine and proline residues and, in this respect, are similar to alveolins (Huttenlauch et al 1995 ; Huttenlauch and Stick 2003 ; Kloetzel et al 2003a , b ; Marrs and Bouck 1992 ). This was confirmed by HHrepID analysis, which readily detected 12-amino acid tandem repeat structures in these proteins (Fig.…”

“…Accordingly, the multi-repeat structures of alveolins are very similar, both in periodicity and amino acid composition, to those of cytoskeletal proteins in other protists: the articulins and plateins. Whilst Euplotes plateins possess canonical ER signal peptides and form plate-like structures inside alveoli (Kloetzel et al 2003b ), Euglena articulins form intermediate filaments with a more classic membrane skeleton role (Huttenlauch et al 1995 ). Our findings give support to the concept that articulins, plateins and alveolins have a common structural scaffold and thus should be grouped within the same protein superfamily.…”

Occurrence of Articulins and Epiplasmins in Protists1

“…FTIR spectroscopic measurement of bacterially expressed Pseudomicrothorax articulin p60 showed about 30%β‐sheet (Huttenlauch et al 1995). An extended conformation would be in line with the filaments that reconstituted from fractions highly enriched in articulins in the presence of plasma membranes (Dubreuil and Bouck 1988) and with observations with purified recombinant articulin that revealed rod‐shaped, short filamentous structures with diameters of 15–20 nm as well as 25–30 nm thick filaments (Huttenlauch et al 1995). Trans‐fection of either articulin 1 or 4 of Pseudomicrothorax into mammalian cells indicated a strong tendency to aggregate to form larger assemblies.…”

Plateins: A Novel Family of Signal Peptide‐Containing Articulins in Euplotid Ciliates1

“…Another protistan cytoskeletal protein featuring many repetitive sequences has been identified and named ‘articulin’. First discovered in Euglena gracilis (Marrs and Bouck 1992) and since described in the ciliate Pseu‐domicrothorax dubius as well (Huttenlauch et al 1995, 1998), articulins are characterized by a core domain of 12‐mer repeats with a consensus alternating valine/proline (VPVPV…) sequence. The extent of this repetitive zone was demonstrated graphically by Marrs and Bouck (1992) using dot‐plots.…”