Analysis of tectonin I and II and tectonin‐related proteins from Physarum polycephalum

Physarum polycephalum , a true slime mold, is a lower eukaryote with a unique life cycle that consists of plasmodium, spore, amoeba, and sclerotium stages. The plasmodia consist of a huge, multinuclear cell that grows without cytokinesis. When the plasmodia are cultured in liquid medium, the cells segregate into numerous small‐walled masses called microplasmodia. The current study focused on the role of tectonin, a lectin‐like protein of the microplasmodia. Microplasmodia express tectonin I and II isoforms. Tectonin II has a larger molecular weight than tectonin I, and the two‐third of amino acid sequence of tectonin II is homologous to the entire sequence of tectonin I. Both tectonin I and II sequences contain six WD repeat motifs that form a beta‐propeller structure, a motif that is generally found in diverse proteins related to signaling processes. Tectonin‐like domains have been found in various types of proteins in a wide range of organisms. Examples include human tectonin (TECPR) and horseshoe crab galactose‐binding protein (GBP). The function of these tectonin‐like proteins has been well studied, whereas that of Physarum tectonin is not well characterized. We have previously reported that tectonin I is expressed during every stage of the Physarum cell cycle. However, haploid‐stage amoebas and spores do not express tectonin II. Therefore, it is believed that tectonin I and II may have distinct functions. Immunofluorescent intracellular localization has shown that tectonin I and II localize to plasmodia cell surface protrusions, suggesting that both proteins may function at the cell membrane and facilitate signal transduction, endocytosis, exocytosis, or other processes. To elucidate the function of tectonin, microplasmodia cell fractionation was performed. Although tectonin I and II were thought to be soluble proteins according to amino acid sequence analysis, both proteins separated into the membrane fraction. Solubilization experiments were performed using detergents, salts, ions, and various buffers to test protein release from the membrane surface. Tectonin I and II were released from the membrane when plasmodia were exposed to an alkaline buffer or to a buffer containing deoxycholate. Next, pull‐down assays were performed to analyze the presence of tectonin‐associated proteins using GST‐tectonin I and II followed by immunoprecipitation using anti‐tectonin I and II antibodies, respectively. Only a few proteins that putatively interacted with tectonin I and II were found. Finally, we confirmed that microplasmodia secrete tectonin‐like proteins in the culture medium during cultivation. We will present preliminary analyses of how tectonin interacts with tectonin‐related proteins.