Association of RABL6 withAP3 in trafficking membrane

Sammanfattning: Via the membrane trafficking system, proteins and macromolecules dispense in different pathways into cells. Moreover, it transports proteins inside and outside of cells. AP3 and LAMP1 are crucial in the biogenesis of lysosomes and sorting the cargo proteins in the trafficking membrane. The result of immunoprecipitation and mass spectrometry proteomics that Paul Manna carried out showed that the RABL6 protein has a relation to AP3. RABL6 has been known as a proto-oncogene previously, and this is the first time that RABL6 may have a function in trafficking membrane. This study aimed to find the association of RABL6 with AP3 and the function of RABL6 via the distribution of LAMP1. HeLa cell line was transfected by a plasmid containing RABL6/GFP. The cells transfected by RABL6/GFP plasmids and wild-type cells were fixed and prepared for immunostaining. After immunostaining, confocal microscopy was used to show the interaction of AP3 and RABL6 and the distribution of LAMP1. In addition, HeLa cells were knockout for RABL6 to show the function of AP3 in RABL6 knockout cells. Optimizing immunostaining, the ratio of 1:100 for RABL6 antibody with PFA fixing shows the best result. The colocalization of RABL6 and AP3 was calculated in cells transfected by RABL6/GFP plasmid and wild-type cells and interpreted the association to each other. The knockout RABL6 cells were unsuccessful, although some differences were observed in the size of the cells. The distribution of LAMP1 in wild-type cells and RABL6 cells transfected by RABL6/GFP plasmid displayed statistical differences. In overexpressed cells, LAMP1 showed more intensity. In conclusion, the RABL6 is involved in trafficking membrane with AP3 and LAMP1.