Protein O-Glycosylation Checkpoint

Every process in a living cell requires a precise control over the localization and the stability of proteins. This requirement is accomplished by a plethora of known and still unknown mechanisms that regulate protein fate. After their biosynthesis in ribosomes many proteins are modified, for instance by attachment of sugars of sugar groups. This process is known as protein glycosylation. In concert with other cellular factors, post- translational modifications often act as address tags to target proteins to their required destination or the determine their molecular shelf life. A subgroup of glycosylation processes called O-glycosylation involves the attachment of sugars to oxygen residues on proteins. Despite hundreds of proteins are O-glycosylated in every higher organism relatively little is known about the function of this modification. In particular, the functional role of the plant specific O-glycosylation of the amino acid hydroxyproline remains largely elusive. This project focuses on the hypothetical role of hydroxyproline O-glycosylation for protein fate control. Recently, genetic defects in transferring galactose to hydroxyproline led to abnormal root growth and seed development and salt intolerance, a phenotype that perfectly mirrors the loss of the FASCICLIN LIKE ARABINOGALACTAN PROTEIN 4 (FLA4) gene. Recent research in my group and by others suggested that FLA4 carries O-glycans and therefore FLA4 provides an excellent paradigm for the function of O-glycosylation of a specific protein in an intact organism. In fact, initial data show that the O-glycosylation machinery influences both the amount and the localization of FLA4. Therefore the working hypothesis of this project suggests that the O-glycosylation of FLA4 and other proteins might be crucial for their cellular fate and hence their function. In a small team I want to address the following questions: In which organelles does the cell sense the degree of protein O-glycosylation? How is protein fate determined according to varying degrees of O-glycosylation? Which genetic and biochemical factors are crucial in this process? Using transgenic plants harbouring functional fluorescent FLA4 in the model plant Arabidopsis thaliana we will utilize O-glycan defective mutants as well as chemical and biochemical inhibitors to define the site and mechanism of protein fate control and to identify novel genetic regulators of this process. New insights into the fundamental cellular processes studied in this project will further extend our understanding of growth and development, and stress tolerance of natural and cultured plant species, preparing human society for future challenges of world population and climate change.

Proteins are a crucial part of every living organism. Each protein needs to be present in the right place and at the right time within a cell. When proteins are no longer needed or become damaged, they are disposed of by the cell. Understanding how the fate of individual proteins is regulated is essential to grasping basic life processes. Many plant proteins have sugar molecule markers called protein glycosylations, whose functions are often not well studied. One type of glycosylation, which is very common but not well understood, is found on Arabinogalactan Proteins (AGPs). Our project investigates whether AGP glycosylations might be used to mark proteins for their disposal or reabsorption by the cell. We focused on a specific AGP called FLA4, which affects root growth and seed development. We discovered that glycosylations at a specific site on FLA4 are necessary for its normal fate, and surprisingly, different types of glycosylations can work for this purpose. We identified two genes and a group of protein-degrading enzymes involved in the cellular processes that contribute to protein disposal depending on glycosylation. Interestingly, many other mechanisms involved in the disposal of different proteins are not important for the fate of FLA4. Our work reveals a new fundamental mechanism for protein control in plants that depends on AGP glycosylation. Insights into this area will contribute to a better understanding of plant growth.