Billions of years ago, the first eukaryotic common ancestor split from its archaeal lineage with a distinct set of genes, the majority of which many of us (eukaryotes) contain to this day. Recently, a high-throughput sequencing approach to identify the essential genes in an archaeal model organism, Sulfolobus islandicus, discovered only 80 genes conserved between present-day archaeal and eukaryotic organisms to be essential in both domains of life. Of these 80, a single has remained poorly characterized: ZPR1.
Previous efforts to elucidate the function of Zpr1 in eukaryotes spanned nearly three decades and biological roles ascribed to Zpr1 varied from cell signaling to splicing. However, none of these roles convincingly painted a picture of the core essential function of Zpr1 in cells, as organisms that required Zpr1 to live lacked the specific signaling receptors and splicing proteins linked to Zpr1’s function in human cell lines. Despite early focus on unconserved Zpr1 interactors, it was eventually found that Zpr1 interacted with a protein whose lineage dated back to the root of the tree of life: translation elongation factor 1A (EF1A). EF1A plays arguably one of the most important roles in the cell: it delivers amino acid-charged tRNAs to the ribosome for nascent polypeptide chain elongation. Consistent with this role, EF1A is one of the most abundant proteins in cells. Despite this link, the function of Zpr1’s interaction with eEF1A in these early studies was suggested to be dispensable.
So why was our lab interested in a protein that had been rendered a blip on the radar since 2007? A key insight came from studies of protein homeostasis (proteostasis) in the workhorse model organism Saccharomyces cerevisiae. A search for the top genomic targets of Hsf1, the master transcriptional regulator of chaperones (protein folders), revealed the ZPR1 locus to be second only to HSP70, a highly conserved, promiscuous, and abundant class of chaperones (Solís et al., 2016, Pincus et al., 2018). Furthermore, our lab later found that rapid depletion of Zpr1 in S. cerevisiae triggered a protein folding stress response equal in strength to depletion of the top class of chaperones, strongly suggesting protein misfolding to be the root of the problem in cells lacking Zpr1.
Eventually, our lab discovered other key observations: newly synthesized eEF1A aggregates and is rapidly degraded by the proteasome in Zpr1-depleted cells. Synthesizing this information, we hypothesized that Zpr1 acts as a bespoke eEF1A chaperone by ensuring that newly synthesized eEF1A is folded into a near-native conformation competent for binding GTP and tRNAs. Our paper demonstrates this point using numerous complementary approaches and finds that Zpr1 is indeed an essential requirement for proper folding of one of the most conserved and abundant proteins in cells. Accordingly, like all other essential genes specific to eukaryotic and archaeal organisms, Zpr1 performs a critical role along the central dogma of molecular biology, providing a satisfying denouement to a long-standing puzzle.
Despite this conclusion, many more questions remain. The discovery of Zpr1’s role highlighted to us a new fundamental challenge faced by eukaryotic cells: proper folding of eEF1A. We have recently identified three more genes in yeast and humans that are all seemingly dedicated to this one folding problem. What aspects of eEF1A’s structure and function drive the need for specific chaperones and cofactors to cater to its folding? As a G protein, eEF1A has an inherent molecular switch timed with GTP binding and hydrolysis. Do the required dynamics of eEF1A’s protein domains, which need to bind and distinguish between a plethora of factors such as tRNAs, condemn eEF1A to needing a specific chaperone? Can we produce a ‘self-foldable’ eEF1A that renders an essential protein (Zpr1) dispensable? What evolutionary tradeoffs exist between requiring a chaperone and being self-foldable? Stay tuned for more.
by Ibrahim (Abe) Sabbarini and Alex McQuown
We Made the Cover!
This cover image evokes the chaperone mechanism by which Zpr1 (‘Zipper 1’) zippers up the domains of newly-synthesized eEF1A (multi-colored strands) into a near-native tertiary conformation (tri-colored ribbon representation). Illustration: Alexa D. Pérez Torres.
