16 Jan 2023
by Richa

Summary- paper 5: Organelle proteomic profiling reveals lysosomal heterogeneity in association with longevity

Yong Yu, Shihong M. Gao, Youchen Guan, Pei-Wen Hu, Qinghao Zhang, Jiaming Liu, Bentian Jing, Qian Zhao, David M Sabatini, Monther Abu-Remaileh, Sung Yun Jung, Meng C. Wang

Biorxiv, 2022

Questions/gaps addressed:

  • Lysosomes are the key platform to modulate the activities of mTORC1 and AMPK signaling, both of which regulate longevity. How does the composition of lysosomes change as their activities modulate or vary with ageing?

Key methods:

  • Rapid Lysosome IP, using 3HA tagged TMEM192 transgenic C.elegans (in whole worms, or tissue specific expression in epidermis, muscle, intestine, and neurons),followed by large-scale proteomic profiling.

  • Comparison of lysosome-enriched proteome with LAMP1/LMP-1 (LMP1-RFP-3HA) Lyso-Tag and the one with Cystinosin/CTNS-1 (CTNS-RFP-3HA) Lyso-Tag.

  • in vitro staining of isolated lysosomes with Lysotracker to monitor the intactness of isolated lysosomes

  • RNAi screen in worms based on LysoSensor fluorescence intensity.

Major takeaways:

  • LAMP1 LysoIP: 216 lysosome-enriched candidates identified, 178 candidates have mammalian homologs. No CTNS in this set.

  • LAMP1 LysoIP in long lived worms: lipl-4 (lysosomal acid lipase) and daf-2 (insulin/IGF-1 receptor): significant number (vATPase, autophagy, mTOR, AMPK related) enriched in mutant (lipl-4) vs wt. Also Nucleoporins (lysosomes also more perinuclear in lipl-4). More mature lysosomes accumulate in mutants?

  • CTNS LysoIP (supplement 6) vs LAMP1 LysoIP: 95 common proteins, more mature lysosomes in CTNS IP.

  • RNAi KD of CTNS and LAMP1 common (95) proteins, followed by lysosomal staining by LysoSensor. vATPAse mutant reduced lysosomal number, increased size. One new effector- TMEM144- predicted carbohydrate transporter, mutant increases lyso pH.

  • Heterogeneity of lysosomal protein composition associated with lysosomal status, tissue specificity, and organism longevity.