LEAPS-MPS: Development of tools to label and isolate nuclear and cytoplasmic O-fucosylated proteins

In this project, funded by the MPS-LEAPS (Launching Early-Career Academic Pathways) Program and managed by the Division of Chemistry (CHE), the Joiner Lab at St. Olaf College aims to develop tools to isolate and study O-fucosylated proteins in plants. O-fucosylation, or the transfer of a single fucose sugar to nuclear and cytoplasmic proteins, is a newly discovered, essential protein modification catalyzed by the SPINDLY (SPY) enzyme. SPY is critical for proper plant growth and development, regulating the plant’s response to environmental signals. Due to a lack of tools available to label and isolate SPY-specific O-fucosylated proteins, there is limited information on SPY’s overall biological function. To address this, Professor Joiner and her students will develop two labeling strategies to tag and isolate SPY-specific O-fucosylated proteins. These strategies will enable chemical or enzymatic labeling to attach a functional handle to O-fucosylated proteins that can be used for their isolation and characterization. This will expand the current toolbox for studying the essential SPY enzyme and its biological function in plants. In addition, this research will increase access for St. Olaf students to interdisciplinary science, training and mentoring them to be independent chemical biology researchers.

Professor Joiner and her students will develop two labeling strategies to isolate and study nucleocytoplasmic O-fucosylated proteins in plants. While tools are available for labeling large, glycan structures on the outside of cells containing O-fucose, there are no tools to label single intracellular O-fucosylated proteins. This limits mechanistic studies of O-fucosylation and the SPY glycosyltransferase that catalyzes the reaction. The first strategy will use bioorthogonal GDP-fucose analogs that SPY will be able to use to transfer the modified fucose to proteins and click chemistry to add a functional handle to these proteins for isolation and characterization. The second strategy will use enzymatic labeling to add a modified sugar to SPY-specific O-fucosylated proteins for chemical functionalization using bioorthogonal probes. Both strategies use bioorthogonal chemistry to enable the efficient and selective isolation of SPY-specific O-fucosylated proteins both in vitro and in vivo, expanding the toolbox for mechanistic studies into the catalytic and substrate selection mechanisms of SPY enzymes and the role of intracellular O-fucosylation in plants and other SPY-containing species.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Award Number: 2531169
Principal Investigator: Cassandra Joiner
Funds Obligated: $199,230
State: MN