Novel Drivers of Eukaryotic Protein Biogenesis and Complex Assembly

The cell’s capacity for protein folding can be controlled by transcriptional regulation of genes encoding chaperones. These factors recognize unfolded proteins to shield them from aggregation or actively assist their folding process. Protein synthesis is an intrinsic folding burden on the cell that is monitored by the heat shock (transcription) factor 1 (Hsf1) to maintain the appropriate expression of a small number of essential chaperones for general protein folding. The starting point for my first body of work was the lab’s observation that Hsf1 in the yeast S. cerevisiae additionally controls expression of Zpr1, a protein that bears no homology to other chaperones but is essential and conserved across eukaryotes and archaea. Using complementary approaches, including biochemical reconstitution and structure-guided mutagenesis, we found that Zpr1 is a chaperone tailored to the final steps in the biogenesis of eukaryotic translation elongation factor 1A (eEF1A), a highly abundant GTP-binding (G) protein comprising ~5% of the proteome. The extreme fragility of eEF1A’s tertiary structure had been historically appreciated since the 1970s, when eEF1A’s biochemical activity was first characterized. Our work explained how cells efficiently solve this problem to enable rapid growth while staving off the inherent potential of abundant eEF1A folding intermediates to disrupt global protein folding in the cell.

My subsequent work was centered on the identification of two additional factors in yeast, both conserved but uncharacterized, that support eEF1A biogenesis. First, we identified Aim29 by forward genetic screening as a factor that promotes Zpr1’s essential function in the cell. Follow-up work by other lab members showed that Aim29 is a co-chaperone for Zpr1 that facilitates substrate release once eEF1A has acquired the ability to hydrolyze GTP. Second, using an AlphaFold-guided computational screen, we predicted the function of Ypl225w as a chaperone dedicated to folding eEF1A’s N-terminal G domain. Using a myriad of assays, we found that Ypl225w associates with ribosomes in the act of synthesizing short eEF1A nascent chains. Ypl225w then remains stably bound to translating ribosomes until the emergence of the full complement of GTP-binding sequence elements within the G domain of eEF1A. Lastly, GTP binding to nascent eEF1A drives G domain folding while triggering release of Ypl225w, thereby allowing chaperone recycling. Together, this body of my thesis work revealed that cells use a dedicated team of folding factors to guide eEF1A’s biogenesis beginning with its nascency on the ribosome. By contrast to general chaperone ATPases, eEF1A ATP-independent chaperones receive cues about biochemical directionality and folding product quality via GTP binding and hydrolysis of their sole client.

Next, we focused on identification of an assembly factor for the eukaryotic chaperonin TRiC/CCT. Chaperonins are large, ring-shaped complexes that mediate folding of proteins inside their nanocages. The cytoskeletal protein tubulin is an obligate CCT folding substrate that undergoes a series of sequential folding steps while being encapsulated. Each of these steps comprises a stereotypical interaction with one of eight paralogous CCT subunits that exist in a defined spatial arrangement relative to one another. Since individual CCT subunits lack inherent information for correct self-assembly, we used AlphaFold-guided computational screening to identify the missing assembly factor(s). We show that a conserved tubulin-like protein (Dml1 in yeast) functions as a CCT substrate mimic to guide the association of the positively-charged half-ring of subunits, namely Cct6, Cct3, and Cct1. Using an electrostatic hook, Dml1 next links Cct1 to the negatively-charged half-ring of subunits via Cct4, which arrives at this junction associated with subunits Cct2, Cct5, and Cct7. Finally, we show that Dml1 is required for the association of Cct8 to Cct6, marking the closing of the chaperonin rings en route to CCT complex maturation.