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Title: Elucidating the function of STX19 and SNAP29 in post-Golgi trafficking
Author: Ampah, Khamal Kwesi
ISNI:       0000 0004 5992 1760
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2016
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There has been a significant amount of research performed to identify and characterise the molecular machinery involved in vesicle transport in eukaryotic cells. This has lead to the identification of several important protein families including coat proteins, small GTPases and SNAREs. There are 38 SNAREs in the human genome and they are localised to the membranes of the biosynthetic and endocytic pathways where they play a role in membrane fusion. The aim of my thesis was to elucidate the function of two poorly characterised Q-SNAREs, SNAP29 and STX19, in post-Golgi trafficking. I have set out to determine where they are localised within the cell; how they are targeted to membranes and to identify which pathways they function on. Using in-house generated rabbit polyclonal antibodies, I have identified a pool of STX19 and SNAP29 that colocalise with tubular recycling endosomal markers, MICAL-L1, Rab8, PACSIN-2 and EHD1. This localisation data indicates that SNAP29 and STX19 have a role in endocytic trafficking. I have shown that STX19's palmitoylation is necessary and sufficient for targeting it to tubular recycling endosomes. I have also determined that MICAL-L1 regulates the recruitment of SNAP29 on tubular recycling endosomes. To gain an insight into the pathways on which STX19 functions I have used an RNAi-based approach. Depletion of STX19 causes the loss of MICAL-L1, RAB8 and SNAP29 from tubular recycling endosomes. This, in turn, leads to the accumulation of the TF-R, GLUT1 and internalised integrins indicating that STX19 has an important role in endocytic recycling. To identify novel molecules which co-ordinate and regulate STX19 function I have used Bio-ID based proteomics and yeast two-hybrid screening. My data indicates that STX19 directly interacts with SNAPs 23, 25 and 29; VAMPs 3, 7 and 8; STXBPs 1, 2 and 5 which validates these approaches. I have also identified several novel interacting proteins including MACF1 and DST that suggest that STX19 may have novel links to the cytoskeleton and integrin trafficking. The Bio-ID based approach has also identified potential cargo molecules which traffic via the STX19 pathway. This list included proteins such as NDGR1, ERBB2IP, VANGL1 and SCRIB that have been shown to be involved in regulating cell migration and cell polarity. Taken together, my data suggests that a pool of STX19 is playing an important role in endocytic recycling and may be required for regulating cell motility and epithelial polarisation.
Supervisor: Peden, Andrew Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available