Molecular interaction between human tumor marker protein p150, the largest subunit of eIF3, and intermediate filament protein K7.

The human tumor marker protein p150 was identified as the largest subunit of eukaryotic translation initiation factor 3 (eIF3) (also known as p170/p180). Its expression level is not only upregulated in many transformed cell lines, but also in several human cancers including breast, cervical, esophageal, and stomach carcinomas. The function of p150 in cancer and initiation of translation are not well understood. Using the yeast two-hybrid genetic screen, we found that a portion of p150 interacts with hPrt1, another subunit of eIF3, and cytokeratin 7, an intermediate filament protein. The interactions between p150 and hPrt1, and between p150 and cytokeratin 7 were verified both in vivo and in vitro. The interaction site for hPrt1 was mapped to the carboxyl half of the coiled-coil region of the p150 protein between amino acids 664-835. The expression of hPrt1 was clearly upregulated in cancer tissue, similarly to that of p150. By contrast, no substantial difference in the expression level of cytokeratin 7 was observed between cancer and normal breast tissue, suggesting that cytokeratin 7 expression is not co-regulated with p150. Taken together, our studies suggest a new role for p150 in translation initiation, possibly by acting as an adapter molecule between the translation initiation apparatus and the cytoskeleton structure in the cell.

Supramolecular structure of a new family of circular proteoglycans mediating cell adhesion in sponges.

Aggregationfactors are the molecules responsible for species-specific cell adhesion in sponges. Here, we present the structure of the aggregation factor from the marine sponge Microciona prolifera, which constitutes the first description of a circular proteoglycan. We have analyzed chemically dissociated and enzymatically digested aggregation factor with atomic force microscopy, agarose gel electrophoresis, and Western blots using antibodies against the protein and carbohydrate moieties. Twenty units from each of two N-glycosylated proteins, MAFp3 and MAFp4, form the central ring and radiating arms, respectively, stabilized by a hyaluronidase-sensitive component. MAFp3 carries a 200-kDa glycan involved in homologous self-interactions between aggregation factor molecules, whereas MAFp4 carries a 6-kDa glycan that binds cell surface receptors. A 68-kDa lectin found in cell membranes of several sponge species binds the aggregation factor and its protein-free glycans, as well as chondroitin sulfate and hyaluronan. Here, we show that despite their lack of clear sequence homologies with other known proteoglycan structures, the protein and carbohydrate components of sponge aggregation factors assemble to form a supramolecular complex remarkably similar to classical proteoglycans.

Accumulation in marine sponge grafts of the mRNA encoding the main proteins of the cell adhesion system.

Specific cell adhesion in the marine sponge Microciona prolifera is mediated by an extracellular aggregation factor complex, whose main protein component, termed MAFp3, is highly polymorphic. We have now identified MAFp4, an approximately 400-kDa protein, from the aggregation factor that is translated from the same mRNA as MAFp3. The existence of multiple potential sites for N-glycosylation and calcium binding suggests a direct involvement of MAFp4 in the species-specific aggregation of sponge cells. The deduced partial polypeptide consists of a 16-fold reiterated motif that shows significant similarity to a repeat in an endoglucanase from the symbiontic bacterium Azorhizobium caulinodans and to the intracellular loop of mammalian Na+-Ca2+ exchangers. Restriction fragment length polymorphism analysis indicated that the genomic variability of MAFp4 is high and comparable to that of MAFp3. Their combined polymorphism correlates with allogeneic responses studied in a population of 23 sponge individuals. Peptide mass fingerprinting of tryptic digests of the polymorphic MAFp3 bands observed on polyacrylamide gels after chemical deglycosylation of the Microciona aggregation factor revealed that the variability detected on Southern blots at least partially reflects the individual variability of aggregation factor protein components. Polyclonal antibodies raised against MAFp3 strongly cross-reacted with a 68-kDa protein localized in sponge cell membranes. Immunohistochemical use of the anti-MAFp3 antibodies strongly stained a cell layer along the line of contact in allogeneic grafts. We show that the transcription level of the MAFp3/MAFp4 mRNA in sponge allo- and isografts is clearly increased in comparison with non-grafted tissue. These data are discussed with respect to a possible evolutionary relationship between cell adhesion and histocompatibility systems.

In chromaffin cells, the mammalian Sec1p homologue is a syntaxin 1A-binding protein associated with chromaffin granules.

Membrane proteins of the synaptic vesicle and the presynaptic plasma membrane together with soluble proteins form a secretory fusion complex conserved from yeast to neurons (Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., and Rothman, J. E. (1993) Nature 362, 318-324). Two of the membrane proteins have been localized in chromaffin cells, which secrete catecholamines stored in chromaffin granules. Syntaxin 1A and 1B are found in a plasma membrane-enriched fraction, whereas synaptobrevin is concentrated on the granules. Recombinant syntaxin 1A has been used in an affinity chromatography assay to isolate syntaxin receptor proteins of the chromaffin granules. Solubilized granule membranes contain a single protein with high affinity for syntaxin 1A. Sequencing revealed partial homology with Sec1p, a hydrophilic yeast protein acting late in the secretory process. Genetic suppressor analyses predicted the interaction of Sec1p with Sso1p, a yeast homologue of syntaxin 1A, and with Sec4p, a homologue of rab3A (Aalto, M., Ronne, H., and Keränen, S. (1993) EMBO J. 12, 4095-4104). Although rab3A is present on chromaffin granules, we did not detect it bound to syntaxin 1A together with the mammalian Sec1p homologue (mSec1). The mSec1 peptide sequences are almost identical with respective sequences of a soluble protein, termed Munc-18, reported to be the only brain protein with affinity for recombinant syntaxin 1A (Hata, Y., Slaughter, C. A., and Südhof, T. C. (1993) Nature 366, 347-351). The mSec1/Munc-18 may be a receptor protein for syntaxin 1A on the transmitter vesicles mediating their interaction with the plasma membrane in docking and fusion.