UROP Proceedings 2022-23

School of Science Division of Life Science 12 Division of Life Science Mechanisms of Golgi Membrane Protein Retention Supervisor: BANFIELD, David Karl / LIFS Student: LI, Caifan / BCB Course: UROP2100, Fall Vps74p-based Golgi membrane protein retention mechanism has been an important topic in cell biology studies. Vps74p binds both to the cytoplasmic tail of proteins and the COPI, transporting the mislocalized residents back to Golgi. In former experiments has been completed over the summer, we mimicked the Golgi quantitative retention assay in mammalian cells, which established a chimeric protein reporter with a GOLPH3-recognized tail that was transported to the cell membrane in the absence of GOLPH3 and GOLPH3L genes (which are orthologs of Vps74). But our experiments found that in vps74∆ cells, the chimeric protein reporters with Vps74p-recognized tail are not transported back to the plasma membrane, but instead accumulate in the vacuole. In a subsequent series of experiments, we continued to focus on exploring the reasons for the tendency of chimeric proteins to go to the vacuole and tried to prevent this. This tendency may be related to the preferred TMD length of different organelles or the multiple intracellular pathways to the vacuole. However, our further experiments show that neither elongating TMD length to a more appropriate length of the plasma membrane nor reducing vacuole transporting by blocking the vacuole ALP pathway can localize our chimeric protein reporter to its expected destination, the plasma membrane. Mechanisms of Protein Retention in the Yeast Golgi Supervisor: BANFIELD, David Karl / LIFS Student: LI, Caifan / BCB Course: UROP3100, Spring Vps74p is one of the important proteins in yeast, it binds to the cytoplasmic tail of glycosyltransferases (GTs) and binding groups on the COPI coat to achieve protein retention in the Golgi apparatus and maintain the homeostatic distribution of GTs. GOLPH3 and GOLPH3L are orthologs of Vps74 in humans, the mutations of these genes are closely associated with severe diseases such as cancer, so research into them is of great interest. In this report, we have selected and identified nanobodies (Nb) against GOLPH3 and GOLPH3L using model organisms yeast, with the aim of obtaining information about the binding sites of GOLPH3 and GOLPH3L proteins to other components from these selective nanobodies. Many in vitro and in vivo experiments have been carried out to find these specific nanobodies.