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Systematic Analysis of Asymmetric Partitioning of Yeast Proteome Between Mother and Daughter Cells Reveals “Aging Factors” and Mechanism of Lifespan Asymmetry

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Systematic Analysis of Asymmetric Partitioning of Yeast Proteome Between Mother and Daughter Cells Reveals “Aging Factors” and Mechanism of Lifespan Asymmetry Systematic analysis of asymmetric partitioning of yeast proteome between mother and daughter cells reveals “aging factors” and mechanism of lifespan asymmetry Jing Yanga,b,c,d,1, Mark A. McCormicke,1, Jiashun Zhenga,1, Zhengwei Xiea,b, Mitsuhiro Tsuchiyae, Scott Tsuchiyamae, Hana El-Samada, Qi Ouyangb, Matt Kaeberleinf, Brian K. Kennedye,2, and Hao Lia,b,2 aDepartment of Biochemistry and Biophysics, University of California San Francisco, CA 94143; bState Key Laboratory for Artificial Microstructures and Mesoscopic Physics, School of Physics and Center for Quantitative Biology, Peking University, Beijing 100871, China; cState Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China; dDepartment of Histology and Embryology, Xiangya School of Medicine, Central South University, Changsha 410013, China; eBuck Institute for Research on Aging, Novato, CA 94945; and fDepartment of Pathology, University of Washington, Seattle, WA 98195 Edited by Jasper Rine, University of California, Berkeley, CA, and approved July 27, 2015 (received for review March 30, 2015) Budding yeast divides asymmetrically, giving rise to a mother cell that by natural evolution (9–11); when the accumulation of lifespan- progressively ages and a daughter cell with full lifespan. It is generally limiting damage outpaces the dilution by symmetric cell division, assumed that mother cells retain damaged, lifespan limiting materials keeping the damage to one of the two offspring via asymmetric “ ” ( aging factors ) through asymmetric division. However, the identity partitioning is a general strategy to avoid population senescence. of these aging factors and the mechanisms through which they limit It is generally assumed that budding yeast mother cells retain lifespan remain poorly understood. Using a flow cytometry-based, damaged/lifespan-limiting materials (referred to as “aging factors” high-throughput approach, we quantified the asymmetric partition- hereafter), allowing their daughter cells to reset the clock. Indeed, ing of the yeast proteome between mother and daughter cells during cell division, discovering 74 mother-enriched and 60 daughter- a number of potential aging factors have been reported to accumu- enriched proteins. While daughter-enriched proteins are biased to- late preferentially in mother cells through asymmetric partitioning. ward those needed for bud construction and genome maintenance, One example is extrachromosomal rDNA circles, which are known mother-enriched proteins are biased towards those localized in the to be a limiting factor for lifespan (12, 13) and are retained in plasma membrane and vacuole. Deletion of 23 of the 74 mother- mother cells (14–16). Protein aggregates, carbonylated proteins, and enriched proteins leads to lifespan extension, a fraction that is about reactive oxygen species have also been reported to distribute six times that of the genes picked randomly from the genome. asymmetrically between old mothers and their daughters (17– Among these lifespan-extending genes, three are involved in endo- 22). Preferential retention of membrane transporters in the somal sorting/endosome to vacuole transport, and three are nitrogen mother cells has also been associated with lifespan asymmetry source transporters. Tracking the dynamic expression of specific (23, 24). These observations support the general notion that mother-enriched proteins revealed that their concentration steadily increases in the mother cells as they age, but is kept relatively low in mother cells retain aging factors to themselves, enabling their the daughter cells via asymmetric distribution. Our results suggest daughters to rejuvenate. However, a global view of the identities that some mother-enriched proteins may increase to a concentration of asymmetrically partitioned aging factors and the mechanism that becomes deleterious and lifespan-limiting in aged cells, possibly through which they influence lifespan is still lacking. by upsetting homeostasis or leading to aberrant signaling. Our study provides a comprehensive resource for analyzing asymmetric cell di- Significance vision and aging in yeast, which should also be valuable for under- standing similar phenomena in other organisms. In this work, we took a proteome-centric view to analyze the cell division and lifespan asymmetry between mother and daughter aging | asymmetric cell division | proteome cells in budding yeast. Using a flow cytometry-based, high- throughput approach, we quantified the partitioning of the ellular aging and asymmetric cell division are intimately proteome and identified 74 mother-enriched and 60 daughter- Clinked. In budding yeast, asymmetric cell division yields a enriched proteins. Functional analysis of these proteins suggests mother cell and a daughter cell that are easily distinguishable under mechanisms of asymmetric partitioning at an organelle/ the microscope. Tracking the fate of the mother lineage led to the suborganelle level. We found that mother-enriched proteins are SYSTEMS BIOLOGY discovery that individual mother cells have a finite replicative life- much more likely to becoming aging factors than those proteins span, defined by the number of daughters a mother cell produced chosen at random. The proposed mechanism, as supported by our before senescence (1). It is known that although the mother cell single-cell observations, is that these proteins accumulate in old ages with each division, their daughters retain the same full lifespan mother cells to a high level that becomes lifespan-limiting. Our independent of the age of the mother at least until the last few work sheds new light on the mechanisms of asymmetric cell di- mother cell divisions (2, 3). Thus, the asymmetry in cell division vision and aging. leads to asymmetry of aging. Even in single-celled organisms in which cell division is seemingly Author contributions: J.Y., J.Z., H.E.-S., Q.O., M.K., B.K.K., and H.L. designed research; J.Y., M.A.M., J.Z., Z.X., M.T., and S.T. performed research; J.Y., M.A.M., J.Z., and H.L. ana- morphologically symmetric, such as fission yeast or Escherichia coli, lyzed data; and J.Y., M.A.M., J.Z., B.K.K., and H.L. wrote the paper. asymmetric partitioning of cellular contents can still occur and have The authors declare no conflict of interest. – a differential impact on the aging/deathfateofthetwooffspring(4 This article is a PNAS Direct Submission. 8). Asymmetric cell division is also a general phenomenon in 1J.Y., M.A.M., and J.Z. contributed equally to this work. mammalian cells (e.g., during development or in mitotically active 2To whom correspondence may be addressed. Email: [email protected] or tissues), where cell division typically leads to two cells with distinct [email protected]. fates, often with different replicative potential. It has been argued This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. on theoretical grounds that asymmetric cell division may be favored 1073/pnas.1506054112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1506054112 PNAS | September 22, 2015 | vol. 112 | no. 38 | 11977–11982 Downloaded by guest on October 2, 2021 In this work, we took a proteome-centric approach to explore 130 mins A GFP library Cy5 staining cell division asymmetry and its connection to lifespan asymmetry flow Incubate 170 mins in budding yeast. Using high-throughput proteomics based on a cytometer green fluorescent protein (GFP) library and mother cell labeling, 16 hours we quantified the asymmetric partitioning of the proteome be- tween mother and daughter cells and systematically identified BC mother-enriched and daughter-enriched proteins. Functional an- 3 alyses of these proteins suggest that macrostructures are one basis 3 for asymmetric partition. We found that mother-enriched proteins 2.5 0 2 tend to accumulate in mother cells over time and that the deletion mutants are much more likely to extend lifespan than genes GFP (log10) 2 1 chosen at random, arguing that it is the high concentration that −1 1500 0 limits lifespan. These observations provide a consistent picture of Zscore how asymmetric partitioning of the proteome influences lifespan 1000 −2 −1 asymmetry, and serve as a starting point for generating new hy- −2 potheses on the mechanism of asymmetry and aging. 500 # of Cells −3 −3 Results 0 Mean GFP level of daughter cells (170 mins) −3 −2 −1 0 2 3 4 5 Identification of Mother- and Daughter-Enriched Proteins by Quantifying Cy5 Mean GFP level of mother cells (170 mins) the Asymmetric Partitioning of the Yeast Proteome. To systematically Fig. 1. High-throughput screening for proteins asymmetrically distributed identify proteins that are asymmetrically segregated, we have de- between mother and daughter cells. (A) Schematic of the experimental veloped a high-throughput approach to measure quantitatively the procedure. S. cerevisiae strains from the GFP tagging library were grown in partition of the whole proteome between mother and daughter cells 96-well plates to exponential phase and then stained with Cy5 dye (cells in during cell division. To measure the partition of a GFP-tagged red). Newly budded daughter cells after the initial staining carry little Cy5 protein from the yeast GFP tagging library, we labeled mother cells dye (cells in black) because they do not inherit the cell wall from their with Cy5 and let the population grow for about one generation. The mothers. (B) Example of the flow cytometry data collected from one well at resulting cell population
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