Interaptin, an Actin-binding Protein of the α-Actinin Superfamily in Dictyostelium discoideum, Is Developmentally and cAMP-regulated and Associates with Intracellular Membrane Compartments

Abstract

In a search for novel members of the α-actinin superfamily, a Dictyostelium discoideum genomic library in yeast artificial chromosomes (YAC) was screened under low stringency conditions using the acting-binding domain of the gelation factor as probe. A new locus was identified and 8.6 kb of genomic DNA were sequenced that encompassed the whole abpD gene. The DNA sequence predicts a protein, interaptin, with a calculated molecular mass of 204,300 D that is constituted by an actin-binding domain, a central coiled-coil rod domain and a membrane-associated domain. In Northern blot analyses a cAMP-stimulated transcript of 5.8 kb is expressed at the stage when cell differentiation occurs. Monoclonal antibodies raised against bacterially expressed interaptin polypeptides recognized a 200-kD developmentally and cAMP-regulated protein and a 160-kD constitutively expressed protein in Western blots. In multicellular structures, interaptin appears to be enriched in anterior-like cells which sort to the upper and lower cups during culmination. The protein is located at the nuclear envelope and ER. In mutants deficient in interaptin development is delayed, but the morphology of the mature fruiting bodies appears normal. When starved in suspension abpD − cells form EDTA-stable aggregates, which, in contrast to wild type, dissociate. Based on its domains and location, interaptin constitutes a potential link between intracellular membrane compartments and the actin cytoskeleton.

A novel partner forDictyosteliumfilamin is an α-helical developmentally regulated protein

“…1A). Database searches revealed only weak homologies of the predicted coiled coil domains (19-22% identity, up to 42% similarity) to entries corresponding to proteins known to have α-helical structures, like tail sequences of myosins or Dictyostelium interaptin (Rivero et al, 1998), golgin-245 (Griffith et al, 1997) or the yeast cytoskeletonrelated protein USO1 (Nakajima et al, 1991), indicating a structural analogy between these proteins. Monoclonal antibodies raised against a C-terminally derived polypeptide (amino acids 1612-1907) recognized a single protein of approximately 230 kDa in western blots, which is well in agreement with the predicted molecular mass of FIP (Fig.…”

“…FIP does not contain transmembrane, nuclear localization, or other signal peptide motifs. Database searches for homologous proteins revealed about 20% identity to proteins with coiled coil domains like interaptin (Rivero et al, 1998). The regions of similarity correspond to the proposed coiled coil domains.…”

“…It is interesting to note that several of the homologous coiled coil proteins are associated with membranes. Interaptin, a developmentally regulated member of the spectrin superfamily Journal of Cell Science 117 (21) of actin-binding proteins in D. discoideum with an extended central coiled coil domain, localizes to the cytoplasmic site of the endoplasmic reticulum (Rivero et al, 1998). Golgin-245, originally identified as a member of a family of self-antigens recognized by autoantisera from patients with Sjögrens syndrome (Fritzler et al, 1995;Erlich et al, 1996;Griffith et al, 1997), is a large protein predicted to form extensive α-helical coiled coils and is located at the cytoplasmic face of Golgi membranes and Golgi-derived vesicles.…”

The Dictyostelium discoideum GPHR Ortholog Is an Endoplasmic Reticulum and Golgi Protein with Roles during Development

“…This network was identified in coimmunofluorescence studies as the ER because it overlapped with the ER marker PDI (13). At the nucleus, GPHR-GFP overlapped with interaptin, which is located at the nuclear envelope (23). The ER network emanates from the outer nuclear membrane and extends throughout the cytosol.…”

LINCing the eukaryotic tree of life – towards a broad evolutionary comparison of nucleocytoplasmic bridging complexes

“…Interaptin is an ONM protein of the α-actinin superfamily (Rivero et al, 1998) with a long central coiled-coil domain and a C-terminal domain consisting of a TMD and a short tail ending in -PT, thus similar to the C-terminal PPPT motif of KASH proteins (Figs 1 and 2). The C-terminal domain consisting of the TMD and the KASH-like tail are sufficient for nuclear envelope association (Xiong et al, 2008;Rivero et al, 1998). Interaptin overexpression displaces Sun-1 from the nuclear envelope, which the authors interpret as an interaction that is somewhat different from that of the canonical SUN-KASH complexes.…”