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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 111; no. 39; pp. 14019 - 14026
• Main Authors: Thayer, Nathaniel H., Leverich, Christina K., Fitzgibbon, Matthew P., Nelson, Zara W., Henderson, Kiersten A., Gafken, Philip R., Hsu, Jessica J., Gottschling, Daniel E.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 30.09.2014; National Acad Sciences
• Series: Inaugural Article
• Subjects: Biochemistry; Biological Sciences; Cell Division; Cell membranes; Cytoplasm; Daughter cells; Fluorescence; Gels; Mass spectrometry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Membranes; Microscopy; Mother cells; Peptide Fragments - genetics; Peptide Fragments - metabolism; Proteins; Proteomics - methods; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae - cytology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Saccharomycetales; Stem cells; Yeast; Yeasts; asymmetric cell division; ACD; recombination-induced tag exchange; replicative aging; RITE
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1416079111

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 full-length 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. Significance Long-lived proteins in extracellular spaces (joints/tissues) or within specialized nondividing cells (eye-lens) are associated with age-related decline. However, aging also occurs in dividing stem cells. Although several hypotheses have been proposed to explain how stem cells age, none have addressed whether long-lived proteins contribute to aging, partially because of technical challenges in identifying such proteins. We developed a method to overcome these limitations in the model system Saccharomyces cerevisiae . We identified two classes of long-lived asymmetrically retained proteins (LARPs). Full-length LARPs remain intact throughout the mother cell lifespan and accumulate in abundance or become posttranslationally modified. Fragmented LARPs are original proteins that are partially degraded, yet retained by the mother cell during aging. We speculate that LARPs contribute to the aging process.