Analysis of membrane integration and membrane topology of protein Lunapark.

<p>A. Structure of pro-GLC-TNF, Lunapark-TM1-GLC-TNF, and Lunapark-TM1-G2A-GLC-TNF for analysis of the function of transmembrane domain 1 (TM1) of protein Lunapark. B. cDNAs encoding pro-GLC-TNF, Lunapark-TM1-GLC-TNF were transfected in to COS-1 cells, and their secretion and expression in total cell lysates were evaluated by Western blotting analysis using an anti-TNF antibody. <i>N</i>-glycosylation of the expressed proteins in total cell lysates was determined by the change in its molecular weight after treatment with glycopeptidase F (GPF). S, cell culture supernatant; T, total cell lysates. C. cDNAs encoding Lunapark-TM1-GLC-TNF and Lunapark-TM1-G2A-GLC-TNF were transfected in to COS-1 cells, and their expression and the <i>N</i>-myristoylation of the products in total cell lysates were evaluated by Western blotting analysis and [³H]myristic acid ([³H]Myr) labeling, respectively. <i>N</i>-glycosylation of the expressed protein in total cell lysates was determined by the change in molecular weight after treatment with GPF. D. Structure of Lunapark-TM1-GLC-TNF, Lunapark-TM1/2-GLC-TNF, and Lunapark-GLC-TNF for analysis of the function of transmembrane domain 2 (TM2) of protein Lunapark. E. cDNAs encoding Lunapark-TM1-GLC-TNF, Lunapark-TM1/2-GLC-TNF, and Lunapark-GLC-TNF were transfected in to COS-1 cells, and their expression in total cell lysates was evaluated by Western blotting analysis using an anti-TNF antibody. <i>N</i>-glycosylation of the expressed proteins in total cell lysates was determined by the change in molecular weight after treatment with GPF. F. Schematic representation of the transmembrane topology of protein Lunapark in the ER membrane.