Protein complex formation as a rationale for translation factories

The information encoded in genes in living cells is decoded to produce chains of amino acids called proteins, which fold up to form functional and mature forms. The complement of mature proteins present in a cell dictate its identity, function and health. Proteins carry out most biological functions, catalyzing biochemical reactions as well as serving structural and regulatory roles. Most of these functions involve proteins acting in concert with other proteins, frequently in so-called molecular machines where different protein subunits come together to form complexes. This provides enormous flexibility and control, and cells devote large resources to making these complexes in all organisms from bacteria to man. When it goes wrong and protein complex assembly goes awry, this has wide ranging implications. For example, it is known that protein interaction surfaces are hot-spots for many human genetic disease-associated mutations.

Estimates suggest that in yeast cells there are up to 60 million protein molecules per cell, whereas in a human cell this number rises to over 10 billion, and roughly half of these protein molecules in each system form part of protein complexes. The inherent complexity in producing so many molecular machines raises a key question: how do individual protein molecules within this protein sea find their correct, cognate partner(s) and form appropriate protein complexes? Given that each protein molecule is synthesized independently, using a template molecule, mRNA, it is not immediately obvious how cells solve this 'needle in a haystack' problem. However, recent evidence has shown a simple solution to this challenge is to ensure that the different proteins from the same complex are synthesized in the same defined subcellular location. This can be viewed as a factory, which specializes in making selected protein complexes. In order to achieve this, the cell factory model benefits from colocalizing the template mRNA molecules to the same locale. In this proposal, we will use the yeast cell due to its relative simplicity, and the range of reagents available to resolve a number of key questions linked to this model. We will determine how widespread this factory-associated assembly of protein complexes is, how the factories form and are regulated, and how important the factory-mediated assembly of protein complexes is for a cell's life.

The synthesis and assembly of multi-protein complexes constitutes a substantial cost to a cell's bioeconomy. While protein complexes are ubiquitous, the likelihood that a protein randomly encounters its cognate binding partner in the highly congested eukaryotic cytosol is remote. Failure to interact appropriately leads to increased protein aggregation, a state associated with many diseases. One solution to improve fidelity that also makes energetic sense is to co-translate mRNAs encoding interacting proteins at specific sites in the cytoplasm. Here, protein complexes could fold and assemble, in parallel with the synthesis of individual complex members. This hypothesis provides a compelling rationale to explain recent observations from our lab and others showing that specific functionally-related mRNAs are translated at discrete sites or factories within cells.

Determining the full extent, mechanism and consequences of this process is the focus of this proposal. We will use the model eukaryote yeast to determine the proportion of protein complexes that form cotranslationally. This simple system offers substantial advantages and unparalleled resources, including many mutant and epitope-tagged strain collections and wide-ranging 'omics datasets. Critically, the key eukaryotic translation regulation pathways are conserved in yeast providing an opportunity to establish how cellular stress affects co-translational assembly. For selected newly-discovered and established benchmark protein complexes, we will explore the mechanism by which cotranslational assembly is established including the RNA/ protein sequence elements and factors involved. Finally, we will use a combination of biochemistry, genetics and cell biology to address the ultimate physiological implications of cotranslational assembly. Our work will provide fundamental insights into the process of cytosolic protein complex assembly, generating a framework for future exploration of its role in disease.