Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.818113
Title: Investigating the trafficking of Golgi-resident glycosylation enzymes
Author: Welch, Lawrence
ISNI:       0000 0004 9359 5508
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Abstract:
The Golgi is a major site of glycan processing mediated by a large number of Golgi-resident glycosylation enzymes. These enzymes are largely type II transmembrane domain (TMD) proteins consisting of a short N-terminal cytosolic tail, a short TMD and a lumenal ‘stem’ region which separates the catalytic domain and the lipid bilayer. The cytosolic tail, TMD, and stem together make the CTS domain which is responsible for the Golgi-targeting of these enzymes via multiple COPI-dependent mechanisms. What is not clear is the relative contribution of each mechanism to Golgi retention or how they work to fine tune the sub-Golgi localisation of these enzymes. To explore these nuances of intra-Golgi traffic, a MitoID proteomic screen was applied to the intra-Golgi golgin tethers GMAP-210 and golgin-84 to identify novel intra-Golgi vesicle-resident factors. The screen identified the Rab2 effector of unknown function FAM114A2. FAM114A2 and paralogous protein FAM114A1 were shown to specifically associate with early-Golgi membranes. A predicted ALPS motif was necessary for membrane association, presumably through non-specifically absorbing onto highly-curved membranes. Other regions of the proteins were required to mediate a specific association with membranes of the early-Golgi. GST affinity chromatography and mass spectrometry revealed that FAM114A1 pulled down Rab2A and Rab2B while FAM114A2 pulled down several COPI cargo proteins including many glycosylation enzymes. FAM114A2 interacted directly with the cytoplasmic tails of cargo which had a membrane-proximal polybasic motif. Similar binding studies revealed that the COPI adaptors GOLPH3+3L pulled down the COPI coat and over a hundred novel clients. GOLPH3+3L were shown to interact promiscuously with the cytoplasmic tails of clients with a total predicted charge of ≥4+. An in vivo Golgi retention assay revealed that tails which satisfy the GOLPH3+3L charge threshold are sufficient to impart Golgi-retention in a GOLPH3+3L-dependent manner. The deletion of FAM114A genes had no impact on retention in this assay. The assay also showed that the TMD of ST6GAL1 was sufficient to impart Golgi retention in a GOLPH3+3L-independent manner. The deletion of GOLPH3+3L, but not FAM114A genes, triggered the lysosomal degradation of COPI cargo and caused a global defect in glycosylation in U2OS cells. In contrast, deletion of the D. melanogaster FAM114A ortholog CG9590 caused a defect in mucin-type glycosylation in adult flies. CG9590 interacted directly with the COPI coat and pulled-down a host of COPI cargo proteins including mucin-type glycosylation enzymes. It is likely that FAM114A is an evolutionary-conserved COPI adaptor which acts to recycle a specific subset of COPI cargo from the early-Golgi. In contrast, GOLPH3 proteins are promiscuous COPI adaptors which function as master gatekeepers at the late-Golgi to recycle and retain a broad array of Golgi-residents.
Supervisor: Munro, Sean Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.818113  DOI:
Keywords: Golgi ; COPI ; Glycosylation ; Glycosyltransferase ; Cisternal Maturation ; Sorting Motifs
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