Identification of long-lived proteins retained in cells undergoing repeated asymmetric divisions

Abstract

Long-lived proteins have been implicated in age-associated decline in metazoa, but they have only been identified in extracellular matrices or postmitotic cells. However, the aging process also occurs in dividing cells undergoing repeated asymmetric divisions. It was not clear whether long-lived proteins exist in asymmetrically dividing cells or whether they are involved in aging. Here we identify long-lived proteins in dividing cells during aging using the budding yeast, Saccharomyces cerevisiae. Yeast mother cells undergo a limited number of asymmetric divisions that define replicative lifespan. We used stable-isotope pulse-chase and total proteome mass-spectrometry to identify proteins that were both long-lived and retained in aging mother cells after ∼18 cells divisions. We identified ∼135 proteins that we designate as long-lived asymmetrically retained proteins (LARPS). Surprisingly, the majority of LARPs appeared to be stable fragments of their original fulllength protein. However, 15% of LARPs were full-length proteins and we confirmed several candidates to be long-lived and retained in mother cells by time-lapse microscopy. Some LARPs localized to the plasma membrane and remained robustly in the mother cell upon cell division. Other full-length LARPs were assembled into large cytoplasmic structures that had a strong bias to remain in mother cells. We identified age-associated changes to LARPs that include an increase in their levels during aging because of their continued synthesis, which is not balanced by turnover. Additionally, several LARPs were posttranslationally modified during aging. We suggest that LARPs contribute to age-associated phenotypes and likely exist in other organisms.ACD | asymmetric cell division | RITE | recombination-induced tag exchange | replicative aging

Systematic analysis of asymmetric partitioning of yeast proteome between mother and daughter cells reveals “aging factors” and mechanism of lifespan asymmetry

“…Thayer et al (35) recently used isotope labeling and total proteome MS to identify long-lived asymmetrically retained proteins (LARPs). They identified several full-length proteins (Mrh1, Pma1, Sur7, Thr1, and Hsp26) and many protein fragments.…”

Single cell protein analysis for systems biology

“…Even at the superficial descriptive level, single-cell RNA-seq cannot capture post-translational modifications (PTMs). Well-characterized regulatory mechanisms, such as long-lived proteins [23] and translational regulation [24] make mRNAs poor surrogates for functional proteins. Munsky et al [25] simulated the distributions of mRNA and protein levels for a number of gene regulatory motifs, and showed that depending on the motif, mRNA levels were not necessarily correlated to the corresponding protein’s level.…”

The Upsides and Downsides of Organelle Interconnectivity

“…Pma1 is an ATP-dependent proton pump that generates the essential pH gradient across the plasma membrane, consuming as much as 40% of the cell’s ATP to pump protons from the cytoplasm to the external environment (Capieaux et al, 1989), which in turn facilitates selective transport of a variety of critical molecules into and out of the cell (Cyert and Philpott, 2013). Pma1 has an unusual property: it is a very long lived protein and continues to accumulate at the plasma membrane over the first several divisions of a mother cell’s life (Thayer et al, 2014). The vacuole pH increase appears to be tied to Pma1 by mass action.…”