Investigation into the differential roles of two variants of the adaptor protein 1 (AP-1) complex in protein trafficking

• Grant number: 18/26306-2
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: March 15, 2019
• End date: September 14, 2019
• Field of knowledge: Biological Sciences - Morphology - Cytology and Cell Biology
• Principal Investigator: Luis Lamberti Pinto da Silva
• Grantee: Lucas Alves Tavares
• Supervisor: Margaret Scott Robinson
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: University of Cambridge, England
• Associated to the scholarship: 16/18207-9 - Characterization of protein trafficking pathways involved in lysosome biogenesis and function in human cells, BP.DR

Abstract

The adaptor protein (AP) complexes are heterotetrameric complexes that coordinate protein trafficking in the endocytic and secretory pathways. AP-1, a complex of four distinct subunits (³, ²1, ¼1, and Ã1), is thought to mediate protein trafficking between the trans-Golgi network (TGN) and endosomes though clathrin-coated vesicles (CCV). Specifically, AP-1 has been implicated in the proper trafficking of mannose-6-phosphate receptors (MPRs), which deliver newly synthesized acid hydrolases from the TGN to endosomes for their subsequent transfer to lysosomes. The human genome encodes two isoforms of the ³-adaptin (³1 and ³2) subunit that form two variants of AP-1 (AP-1³1 and AP-1³2). However, the specific role of these AP-1 variants in MPR trafficking is not well described. Our preliminary data indicate that AP-1³1 is most likely involved in the anterograde trafficking of cation-independent (CI)-MPR molecules from the TGN to endosomes, whereas AP-1³2 seems dispensable for this transport route. Instead, our preliminary data suggests that AP-1³2 could be involved in the retrograde transport of CI-MPR back to the TGN. Here we propose to investigate the differential roles of AP-1³1 and AP-1³2 complex in protein trafficking within the endo-lysosomal system. Therefore, we would like to perform immunoelectron microscopy (EM) of HeLa cells to compare the subcellular distribution of AP-1³1 and AP-1³2, as well as determine the subcellular localization of CI-MPR in cells depleted of AP-1³1 or AP-1³2 by RNAi. Moreover, to expand the knowledge of AP-1³1 or AP-1³2 roles in protein trafficking, we propose to 1) identify new binding partners of AP-1³1 and AP-1³2 in Hela cells using the BioID technique followed by mass spectrometry and 2) to compare the protein composition CCV-fractions from control and AP-1³1 or AP-1³2 knocksideways cells. The work proposed here complements my graduate studies funded by FAPESP (2016/18207-9) and will contribute to better understand the function of AP-1³1 and AP-1³2 in protein trafficking. (AU)