Asymmetric protein inheritance in yeast replicative aging
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Abstract
In contemporary research, the utilization of diverse tags like fluorescent proteins or small epitopes, for the investigation of proteins of interest has become ubiquitous. Normalizing the extensive usage of tags has made us overlook the critical question of how each tag influences the functionality, subcellular localization and interactions with protein-protein networks of the respective protein of interest.
In this study I sought to shed light on this topic by systematically analyzing the effect of external tags on different proteins in budding yeast, Saccharomyces cerevisiae.
To achieve this goal, I chose to assess the impact of tag position, at C-terminus or N-terminus, on a given protein. In order to conduct a genome-wide comparison of the tag position effects, I selected the non-fluorescent protein Halo tag. The Halo tag is a commonly used tag that has a similar size to commonly used fluorescent proteins, rendering it a suitable model for extrapolating its effects to tags of similar sizes. Additionally, for distinguishing between C-terminusly and N-terminusly tagged strains, I decided to use different cytosolic markers for each strain type.
I conducted a comparative analysis between ORF-3xMyc-Halo and Halo-3Myc-ORF libraries, which I also constructed, to evaluate the effects of tagging proteins at the C-termius and at the N-terminus. To ensure a fair comparison between the two, I developed a high-throughput yeast competition assay in liquid medium. This study involved competing 5,340 strain pairs over a four-day period, with the identification of C-tagged and N-tagged cells accomplished through cytosolic fluorescence measurements via flow cytometry.
After the genome-wide competition, I noted two significant observations. First, there is a general trend towards enhanced strain fitness when ORFs are tagged at the C-terminus. I identified 292 ORFs that demonstrate improved fitness when tagged at the N-terminus, 4310 ORFs that show no significant difference in fitness based on tag position and 649 ORFs that exhibit enhanced fitness when tagged at the C-terminus. Secondly, essential genes appear to be particularly susceptible to the impact of tagging, in contrast to non-essential genes. This observation aligns with the notion that any disturbance to essential genes has a more detrimental effect.
In this study, I generated a comprehensive genome-wide catalog that identifies the protein terminus at which introducing a tag is less detrimental to the final strain’s fitness.
After this initial finding, I proceeded to leverage this information to investigate replicative aging in budding yeast. Given the organisms' asymmetrical replication, I employed a tandem fluorescent protein timer (tFT) to track proteins with age-dependent subcellular distributions during mitosis. After evaluating various tFT options, I opted to create a library in which 5,561 ORFs were tagged at the C-terminus with the selected tFT, mScarlet-I-mNeonGreen.
Following the establishment of a high-throughput imaging protocol, I conducted imaging of the aforementioned library to obtain protein age data of each protein pool in both the mother and the daughter cells during mitosis. After manually annotating proteins that displayed asymmetrical segregation based on their age, I identified 775 proteins of interest. It is important to emphasize that these hits will require further validation through our automated image analysis pipeline. However, this initial exploration suggests a promising approach for identifying candidate proteins whose age may correlate with cellular damage and, ultimately, the aging of the cell receiving the older protein pool. Completing this analysis will also yield a new catalog of proteins in budding yeast that could be potentially acting as aging factors.