Scaling of cellular proteome with ploidy

Background:

Inside the nucleus of a cell, DNA (the genetic blueprint) is arranged into structures known as chromosomes (1). Most eukaryotes have two copies of each chromosome (i.e are diploid; 2n) or one copy (i.e are haploid; 1n) but in some cells, three of more copies can be found (i.e are polyploid; >2n). For example, polyploids are common in the plant kingdom where they contribute to plant species diversity (2). But why is polyploidy useful? Being polyploid could help species (and cells) by altering patterns of gene expression to adapt in response to stress or when they encounter a new environment (3-5). However paradoxically, polyploidy can be harmful if it originates from a mistake made by the cell (5), for example, polyploidy is commonly found in human cancer cells, arising when errors are made during the division of chromosomes into the two new daughter cells during mitosis (5). Though polyploidy plays a role in cancer, we still don’t understand how these changes in chromosome number affect other processes in the cell, including how protein levels are affected. Very few differences are found at the level of transcription (when DNA is copied to RNA; 6-7) in polyploidy cells even though they often change in appearance (i.e can be larger) and behaviour. Here, the authors ask how changes to the number of chromosomes instead affect the proteins levels in cells (i.e the ‘proteome’). They use budding yeast (Saccharomyces cerevisiae) as the model. Using yeast with different levels of ploidy (from haploid to tetraploid), they show that increasing ploidy does increase the amount of protein however it is not proportional. This effect is linked to the repression of a novel signalling network which acts through the Tor1 kinase (Target Of Rapamycin), reducing the production of rRNA needed for ribosome production. The TOR pathway is a core pathway which regulates the metabolism of a cell (8).

 

Key Findings:

When budding yeast increase in ploidy, cell and nuclear volume increase proportionally but being polyploid is also associated with genome instability, problems regulating the cell cycle and reduced fitness.

1) Quantitative proteomes as a tool to assess protein abundance in polyploid cells

The authors use a technique called SILAC (stable isotope labelling of amino acids in cell culture) to study how ploidy affects the proteome (9). Briefly, SILAC uses two cell populations, one of which is grown in normal culture medium and the other in medium containing heavy isotopes (forms) of certain amino acids, for example heavy carbon (^13-C instead of ^12-C). The populations can be combined, the proteins identified by mass spectrometry, then the two populations separated based on the presence or absence of the heavy or light amino acid isotope. This approach can be used to investigate differences in protein abundance between different samples. Using SILAC, they show that cells do not show a proportional increase in their protein abundance (referred to as ‘ploidy specific protein scaling’; PSS).

2) Some proteins do scale proportionally in polyploid cells.

Though most protein levels do not increase proportionally, the authors found that proteins involved in cell wall integrity did increase and proteins involved in mitochondrial or ribosomal biogenesis decreased. This was not related to the size of the yeast. These proteins undergo ‘polyploidy dependent regulation’ (PDR).

3) Polyploid cells reduce rRNA production.

In polyploid cells, the authors detect a reduction in the level of rRNA, which is a non-coding RNA component of the ribosomes) in keeping with the effects they saw on ‘ribosome biogenesis’ factors. They next asked if the process of translation (when ribosomes produce proteins) was affected in polyploid cells, finding that the translation rate did increase but it was not proportional and that increased ploidy increased cell sensitivity to translation inhibitors.

4) Loss of Tup1 reduces rRNA levels

As rRNA levels reduce it is likely rDNA transcription also decreases. The authors asked what could be repressing this process. They found that a protein called Tup1 (Tle1 in humans), which when deleted in yeast, was associated with reduced rRNA levels. A key pathway involved in regulating ribosome biogenesis is the mTOR (mammalian Target Of Rapamycin) pathway. Investigating this further, they found 1) if they blocked this pathway in haploid cells, the level of Tup1 was similar to the levels found in polyploid cells and, 2) polyploid cells were more sensitive to the inhibitor (Rapamycin) used to inhibit the mTOR pathway. In all, these data suggest a key role for this pathway in regulating protein abundance in polyploid cells.

 5) A new ploidy-associated protein regulatory pathway

During ribosome biogenesis, the kinase Tor1 phosphorylates another kinase called Sch9 (P70-S6K in humans). The authors show, though a combination of complimentary experiments, that deleting Sch9 resulted in increased rRNA and Tup1 levels and they confirm that Tup1 is a target of the Sch9 kinase suggesting these proteins act in a pathway together. In polyploid cells, Tor1 and Sch9 activity is reduced whereas Tup1 accumulates. Here, the authors have uncovered a new pathway which is linked to changes in protein abundance in polyploid cells. They also show a similar pathway operates in human polyploid cells.

 

What I liked about this preprint:

Given how common polyploidy is in the plant kingdom and the correlation between polyploidy and human cancer, we still lack a lot of information as to how being polyploid affects processes within the cell, particularly gene expression patterns are remodelled at the level of the proteome. This study by Yaha and colleagues now provide an explanation as to why increased ploidy does not cause a linear increase in the level of protein abundance. Polyploid cells reduce rDNA transcription via a novel mTOR associated pathway. Though more work is required to understand the complete functionality of this new pathway, the network of factors involved and why specifically polyploid cells use this approach to moderate their proteome, their data has uncovered a key piece of this complex puzzle and will open up investigations as to the role of this pathway in the context of cancer. It will be exciting to see how the authors explore this pathway further.

 

Questions for the Authors:

 

Q1: When you treat haploid cells with Rapamycin, they increase Tup1 abundance. Do ploidy changes begin to emerge in the population of your Rapamycin treated haploids?

Q2: Tor1 and Sch9 activity is reduced in polyploid cells. What process is acting to limit their activity? Do they become less stable?

Q3: You suggest that transcription of the rDNA locus could be down-regulated to prevent or limit R-loop formation and homologous recombination. Do Tup1 deficient cells show increased levels of R-loops mapping to the rDNA locus? Are Tup1 cells defective in homologous recombination?

Q4: What happens to the ‘metabolome’ of polyploid yeast cells?

 

References:

1. https://www.genome.gov/about-genomics/fact-sheets/Chromosomes-Fact-Sheet
2. Pele, M. Rousseau-Gueutin and A-M. Chevre. Speciation success of polyploid plants closely relates to the regulation of meiotic recombination. Frontiers in Plant Science. 2018.
3. Allario, J. Brumos, J.M. Colmenero-Flores, D.J. Iglesias, J.A. Pina, L. Navarro et al. Tetraploid Rangpur lime rootstock increases drought tolerance via enhanced root abscisic acid production. Plant Cell Environment. 2013.
4. Comai. The advantages and disadvantages of being polyploid. Nature Reviews Genetics. 2005.
5. T. Fox, D.E. Soltis, P.S. Soltis, T-L. Ashman and Y. Van der Peer. Polyploidy: A biological force from cells to ecosystems. Trends in Cell Biology. 2020.
6. Storchova, A. Breneman, J. Cande, J. Dunn, K. Burbank, E. O’Toole and D. Pellman. Genome-wide genetic analysis of polyploidy in yeast. Nature. 2006.
7. Y. Wu, P.A. Rolfe, D.K Gifford, G.R. Fink. Control of transcription by cell size. PloS Biology 2010.
8. Kim and K-L. Guan. mTOR as a central hub of nutrient signalling and cell growth. Nature Cell Biology. 2019.
9. E. Ong, B. Blagoev, I. Kratchmarova, D.B. Kristensen, H. Steen, A. Pandey, M. Mann. Stable isotope labelling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Molecular Cell Proteomics. 2002.

Tags: ploidy, proteome, yeast

doi: https://doi.org/10.1242/prelights.29285

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