Abstract
dc:description.abstractNew proteins and lipids are modified by the Golgi apparatus (Golgi) before being sorted for transport to their final destinations. The Golgi comprises sequential membrane-bound, motile compartments called cisternae. The most prominent Golgi modification is glycosylation, the addition of glycans by a group of enzymes called glycosyltransferases. Glycosylation has no template; therefore, it relies on the sequential organisation of glycosyltransferases within the Golgi. The cisternal maturation model proposes that cisternae progress through the Golgi, acquiring the necessary components as required. In theory, this would lead to resident Golgi enzymes becoming mislocalised. One mechanism to prevent enzyme mislocalisation is their capture by vesicles formed by coat protein complex 1 (COP1), via cargo adaptors such as Golgi phosphoprotein 3 (GOLPH3), for recycling to earlier parts of the Golgi. Published genome-wide CRISPR-Cas9 knockout screens interrogating glycosylation pathways have implicated the highly conserved Transmembrane Nine Superfamily Member 2 (TM9SF2) as a potential novel cargo adaptor. Additional evidence supporting this role comes from the observations that loss of TM9SF2 influences the cellular localisation and/or stability of some glycosyltransferases, and that orthologues possess a COPI binding motif. Taking an explorative approach, we generated knockout cell lines for all four members of the human TM9SF family and used assays including flow cytometry, immunofluorescence, immunoblotting, and biochemistry, to reveal novel glycosylation and morphological phenotypes, as well as new interactors. We provide evidence to confirm the cytosolic protein, Synapse-associated protein 1 (SYAP1), is a binding partner of TM9SF2, and hypothesise it may play a role in functional regulation. In addition, we identify two novel TM9SF2 cargo proteins, the enzymes, β-1,4- galactosyltransferase 1 (B4GALT1) and Sulfhydryl oxidase 2 (QSOX2). We show that two residues within the transmembrane domain of B4GALT1 are required for interaction with TM9SF2. Furthermore, as QSOX1, a paralogue of QSOX2, plays a role in regulating the activity of Golgi enzymes, this may suggest an alternative mechanism for TM9SF2 in the regulation of glycosyltransferases, by regulating 4 disulphide bond formation. Our results provide greater context to the role of the TM9SF family, and in particular support the hypothesis that TM9SF2 is a cargo adaptor involved in the retention of resident Golgi enzymes.
Degree
thesis:*- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2025
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Brown, Katherine
- Advisor dc:contributor.advisor
-
- Munro, Sean
Subjects
dc:subject × 6Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.123747
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/393410