These final results indicated that membrane translocation mediated by TM1 and its flanking locations proceeds irrespective of protein N-myristoylation. Therefore, protein N-myristoylation takes place on protein Lunapark is not necessary for membrane translocation and membrane topology formation of protein Lunapark.
Detection of protein N-myristoylation in in vitro-synthesized protein Lunapark by MS analysis. A. Purified in vitro-synthesized protein Lunapark (1 mg) was divided by electrophoresis in an SDS-Web page gel (twelve.five%). B. MALDI-MS of the tryptic peptides from in vitro-synthesized protein Lunapark. The peaks of the tryptic peptides derived from in vitro-synthesized protein Lunapark are indicated by stars. C. MS/MS evaluation was executed for the peak at m/z 846.55. The recognized N-myristoylated N-terminal sequence is revealed.
In order to figure out the function of protein N-myristoylation of protein Lunapark in the intracellular localization of this protein, cDNAs encoding protein Lunapark C-terminally tagged with FLAG tag and its G2A mutant ended up generated, and their intracellular localization was identified by immunofluorescence examination of HEK293 T cells transfected with these cDNAs. As shown in Determine 5A, [3H]myristic acid labeling uncovered that Lunapark-FLAG was efficiently N-myristoylated, whilst incorporation of [3H]myristic acid was not observed on LunaparkG2A-FLAG. Immunofluorescence investigation of HEK293T cells transfected with cDNA encoding Lunapark-FLAG unveiled that Glycyl-L-prolyl-L-arginyl-L-proline acetate overexpressed Lunapark-FLAG showed a attribute distribution pattern with large polygonal tubular constructions, as proven in Determine 5B, a. In contrast, in the scenario of Lunapark-G2A-FLAG, the characteristic distribution pattern with a huge polygonal tubular composition was not observed, as demonstrated in Figure 5B, b. Since a reticular network composition was noticed on the ER, it was speculated from these observations that protein Lunapark especially localized to the ER and induced tubular ER formation in an N-myristoylation-dependent way. In buy to confirm this, result 
of overexpression of protein Lunapark on the distribution of ER localized protein was evaluated. For this experiment, EGFPSec61b was used as common ER marker protein that distributes all through the ER [37]. In HEK293T cells expressing EGFPSec61b, perinuclear localization of EGFP-Sec61b was observed as proven in Determine 6A, b. In this case, peripheral tubular structure was not noticed even in the overexposed black-and-white image (b9). When HEK293T cells expressing EGFP-Sec61b were stained with ER-Tracker Crimson, 23630290EGFP-fluorescence of EGFP-Sec61b totally merged with fluorescence of ER-Tracker Crimson as proven in Determine 6B, j,k,l. These results evidently shown the particular localization of EGFP-Sec61b to the ER. When EGFPSec61b was coexpressed with Lunapark-FLAG, big polygonal framework about the perinuclear sheet was observed with EGFPSec61b (e,e9). In distinction, when EGFP-Sec61b was coexpressed with non-myristoylated LunaparkG2A-FLAG, the attribute distribution pattern with a big polygonal tubular composition was not noticed with EGFPSec61b, as is the situation with the distribution sample of LunaparkG2A-FLAG, as revealed in Figure 6A, g,g9,h,h9.