Quantitative proteomics reveal PARKIN translocation

An asymmetric cell division produces two daughter cells with different cellular fates. This process is very important for the formation and development of cancer and has significant therapeutic potential. 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.

Firstly?we build a system to identify the long-lived proteins. 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).

Next, we take another system to test and verify the long-lived proteins. Tagging a protein of interest with the RITE system creates a fusion protein, which initially expresses protein-GFP, then through an estradiol-inducible recombination event, expresses protein-RFP. The original protein is labeled green, and subsequent proteins are labeled red.

Finally, accumulation of some LARPs with successive cell divisions may result in an effective increase in dosage of protein in older cells. Plasma membranes LARPs can be both modified and increase in levels with successive cell division

Long-lived proteins contribute to age-associated phenotypes and likely exist in other organisms.

The PARKIN E3 UB ligase is mutated in Parkinson’s disease (PD) and controls mitochondrial autophagy. PARKIN functions via a so-called RING-HECT hybrid mechanism wherein a catalytic Cys residue in the RING2 domain receives UB from an E2 and transfers it to substrate. Here we use quantitative proteomics and live-cell imaging to dissect individual steps in the PINK1 kinase-PARKIN UB ligase mitochondrial control pathway abnormal in Parkinson’s disease.

PARKIN activation by PINK1 produces UB chains on mitochondria, and PARKIN-dependent chain assembly is required for accumulation of poly-phospho-UB on mitochondria. A ten-plex tandem mass tagging quantitative proteomic experiment revealed an increase of 3-fold in p-S65 UB at 30 min after depolarization in PINK1+/+ mouse embryonic?broblasts (MEFs).

PINK1 promotes PARKIN association with poly-UB chains by phosphorylating both PARKIN and poly-UB.

UB can undergo chain extension on seven lysines and the N-terminal methionine, with different fates for different linkage types. PARKIN promotes the synthesis of canonical and non-canonical poly-UB chains on depolarized mitochondria in a manner that depends on its catalytic Cys residue and S65 within its UBL domain.

To summary, we take the power of proteomics to analysis individual steps in complex phosphorylation-driven UB cascades. It will be useful for understanding signaling-ubiquitylation pathways in the future.

Numerologist Warda is hooked on OG-L002 fishing, collecting. And lastly her encouragement comes from socializing along with her companions.