Dissecting protein quality control mechanisms of mislocalized proteins
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Abstract
Protein homeostasis plays an important role in maintaining a healthy proteome within the cell with its quality control systems. The breakdown of protein homeostasis leads to the accumulation of mutated, misfolded, damaged, and mislocalized proteins, many of which are associated with various diseases. Mislocalized proteins are proteins that fail to reach their native compartment or assemble into their native complexes. Mislocalization can occur due to intrinsic inefficiencies of targeting, folding, trafficking, or complex assembly. Various diseases have been associated with or arise due to mislocalization of proteins, and furthermore, the extent of mislocalization increases with aging and in aneuploidy. It is therefore important to better understand the cellular mechanisms that handle mislocalized proteins.
To this end, I sought to systematically identify proteins that are degraded upon mislocalization and then dissect and characterize the properties of these proteins. In order to accomplish this, I established a proteome-wide platform to study the fate of mislocalized proteins upon overexpression of individual proteins using a tandem fluorescent protein timer tag. The tag provides information on protein abundance and turnover. Using this approach, I screened 4211 proteins for their behaviour upon overexpression at different levels using low and high copy plasmids with endogenous and GAL1 promoters. My results show that ~15% of proteins are attenuated upon overexpression with a 2µ plasmid with endogenous promoters. These proteins are distinct from proteins that were attenuated in aneuploidy conditions. In addition, the main characteristics of the attenuated proteins are that they are enriched for subunits of complexes and essential genes, are highly synthesized, and have long half-lives. Aside from the 15% of attenuated proteins, I also found that another ~8% of proteins were destabilized but not attenuated upon overexpression. I showed evidence from the data and the protein properties that support my hypothesis for various methods that the cell may be using to manage and deal with protein mislocalization upon overexpression, depending on the protein’s change in protein levels and stability.
I am able to show that the data also recapitulates the known degradation of cytosolic ribosomal proteins upon overexpression, however, the mitochondrial ribosomal proteins lack any attenuation. In fact, there was a depletion of mitochondrial proteins from the attenuated proteins. Only with an even stronger overexpression, I see attenuation of more mitochondrial proteins but still very minimal for the mitochondrial ribosomal proteins. I also showed that in general, the tagged mitochondrial proteins still localize correctly upon overexpression of an untagged version of the protein. The lack of attenuation of mitochondrial proteins did not change with increased growth temperature or on non-fermentable media.
Focusing on the subunits of complexes, I found that not all subunits within a complex were similarly attenuated. This difference in attenuation behaviour is likely not due to synthesis rates or half-lives, but rather, presence of chaperones or direct interaction partners, assembly factors, and assembly sequence of a complex. Through overexpressing and knocking out every partner subunit within the ER membrane complex, I was able to dissect and propose an assembly model for this complex with the information on its structure. I tested several other complexes but not all were clear and easily interpretable.
Based on my research, I present a large dataset of proteins that are attenuated upon overexpression which can be used to further dissect the protein quality control mechanisms that deal with mislocalized proteins. My research paves the way for further research into the proteins that are attenuated and/or destabilized upon overexpression and the likely mechanisms that are in play for each. I also present a method to dissect the assembly sequence of a complex and subunit pairs that are interdependent for stability.