Using proximity labelling and mass spectrometry to identify novel interactors of the ESCRT-I protein UBAP1 and the accessory protein HDPTP

Abstract

Endosomal sorting complexes required for transport (ESCRTs) coordinate reverse topology membrane remodelling events. The ESCRT-I component Ubiquitin-associated protein 1 (UBAP1) and ESCRT scaffold His domain protein tyrosine phosphatase (HDPTP) are essential for the trafficking of internalised epidermal growth factor receptor (EGFR) to the lysosome via the multivesicular body. HDPTP coordinates numerous ESCRT members at the endosome through their binding to several sites within the HDPTP N-terminal region, including the Bro1 domain, coiled-coil domain and proline rich region. However, little is known about the function of the HDPTP C terminal region despite numerous mutations within this region resulting in disease. UBAP1 contains ubiquitin binding domains but it is still unclear whether UBAP1 binds ubiquitinated cargo, and as such, its function is still largely unknown. Mutations in UBAP1 result in hereditary spastic paraplegia but the mechanism underlying the disease remains elusive. Proximity labelling and mass spectrometry were selected to identify novel interactors which will further expand our knowledge of HDPTP’s and UBAP1’s roles in receptor trafficking and how their dysregulation might result in disease. CRISPR/cas9 knock-in failed to generate cell lines expressing endogenous HDPTP tagged with UltraID. However, HeLa cells were transduced with lentivirus containing UBAP1 fused to the biotin ligase UltraID (UID). Exogenous UBAP1 was expressed at close to endogenous levels, incorporated into the ESCRT-I complex efficiently and rescued the phenotype that occurs upon knockdown of endogenous UBAP1. Mass spectrometry identified biotinylated proteins from UBAP1-UID expressing cells pulled down on streptavidin beads. A control construct with a self-cleaving P2AT2A site situated between UBAP1 and UltraID identified non-specific hits. Epidermal growth factor (EGF)-triggered internalisation of the model receptor, EGFR, was used to identify receptor trafficking-specific interactors of UBAP1. UBAP1-UID was also employed in cells depleted of the downstream ESCRT component VPS4, which traps ESCRTs and associated factors at the endosome, to further identify near-neighbours of UBAP1.

Date of Award	15 May 2025
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Philip Woodman (Main Supervisor), Viki Allan (Co Supervisor) & Martin Lowe (Co Supervisor)