Asymmetrically inherited multidrug resistance

transporters are recessive determinants in cellular

replicative ageing
Amr Eldakak, Giulia Rancati, Boris Robinstein, Parma Paul, Veronica Conway, Rong Li

By
Anubhav
 Introduction
• Ageing is the accumulation of changes in an
organism or object over time.
• Physical, Psychological, and Social change.
Yeast ageing
• 1.Enlargement and fragmentation of nucleolus.
• 2.Damaging of protein due to oxidation.
• 3. Many others
 Enlargement and fragmentation of nucleolus
• rDNA
• Position chromosome no XII,
• Consist directly repeat of 200 copies, each repeat
have own ARS.
• Active part : ribosomal RNA
• Silent part
• Silencing due to Sir2 silencing protein complex.

Young Old
 Protein damaging by oxidation
• Reactive oxygen species (ROS)
• Main source : Mitochondria
• Cause interfering in metabolism*

Old fraction of cells

Young fraction of cells

*Peter laun et al 2001molecular microbiology

• Asymmetry In age
• Buds are free of these deleterious materials.

*Henderson et al 2008, current opinion in cell biology

What are the recessive factors for ageing ?
 MDR proteins transporter

• MDR proteins have roles in cellular

metabolism , detoxification and stress
response.
• Newly synthesized transporter proteins are
mostly deposited in to the bud during division.
Survey of the group of plasma proteins that
belongs to the MDR protein families !*

*Ernest et al, 2005 Methods enzymology

*Huh et al, 2003 Nature
MDR transporters asymmetrically localizing to the mother cell
during polarized growth
Protein Function
Ctr1 High affinity copper transporter
Fet3 Oxidoreductase required for high affinity iron uptake
Fui 1 High affinty uridine permease
Hip1 High affinity histidine permease
Hnm1 Choline/ Ethalonamine transporter
Mrh1 Membrane protein similar to Hasp 30 and Yro2
Pdr5 Multidrug ABC transporter
Pdr12 Multidrug ABC transporter
Snq2 Multidrug ABC transporter
Tat1 Amino acid transporter
Tpo1 Polyamine transporter
Vht1 High affinity vitamin H Symporter
Yor1 Multidrug ABC transporter
Yor2 Stress- related transporter of unknown function
Localization pattern of different proteins
Percentage of localization of protein
at different stage of cell cycle
 Tpo1 localization Pattern during the cell cycle

•Tpo1 GFP deposition during anaphase was strongly biased

Fluorescence intensity of Tpo1-GFP on the cortex of the
mother cell and in the daughter cell
Bud
Mother
> Analysis of Microarray data from yeast genome database*
>Majority of MDR proteins show peak mRNA levels at
metaphase

Quantitative RT- PCR using budding index nuclear morphology

SWI 5 as metaphase marker
When protein is migrate to bud ?
Pulse expression of Tpo1- GFP at
different cell cycle
 Why the old Tpo1 protein
migration does not take place ?
1.Septin mutants cdc12-6 (temp sensitive)
grown at 37 C°.
2.Frap analysis of yeast plasma membrane
 Inference
• Mother cell keeps its own pool of MDR proteins.
• Daughter cell inherits the majority of the newly
synthesized MDR proteins during mitosis.
• Daughter cell receive most of the MDR proteins
at its birth.
• Ageing MDR protein population is retained in the
mother cell and restricted from entering the bud.
What is the rate of decay of MDR
proteins ?
• Modeled the dynamics of the MDR proteins
protein population over a yeast cells RLS.
• The model assume deposition of newly
synthesized MDR protein at a cell birth.
• To estimate decay rate α Tpo1 expression was
induced from the GAL1 promoter (3h) and
then repressed with glucose.
• Stability was monitored by FACS continued…….
• Tpo1 satbility and decay through out the
replicative life span
 Result of FACS analysis

• Rate of decay was observed α =0.16.

Is that Tpo1 activity changes as cell become old?

• Radioactive Spermidine uptake activity was
measured by old and young cells fraction

Spermidine uptake, for equal dry weight Young and Old

Yes! Activity of Tpo1 is decreases as cell become
old.

What is the relation between Tpo1 level and

Tpo1 activity ?
MDR proteins have any function in the RLS?
• Micro manipulation assay to measure the RLS of
mutant cells for..
• TPO1
• YOR1
• CTR1
 Alternative way to test MDR
transporter limiting factor for RLS
• By increased MDR protein level.
• Extra copy of the
• TPO1
• YOR1
• CTR1
Comparative graph of RLS when addition and deletion of
gene was done

Deletion is more severe in

comparison to addition of gene
 Summary
• MDR proteins exhibit a unique asymmetric inheritance
pattern as a result of their timed expression in the cell cycle.
• This suggest that these proteins are marker of cellular
replicative ageing.
• Tpo1 decline in the transporter activity during RLS
• This suggest that these proteins are ageing determinant.
• In addition to deleterious , dominant ageing factors MDR
proteins represents a class of recessive factors, beneficial
factor that limits ageing .
 Future study
• What are the relevant substrate of MDR transporters that
affect ageing
• How changes in the level of proteins interact with other well
studied pathways of cellular replicative ageing.
• Several mammalian ABC transporters are well known stem-
cell markers and are down regulated at the time of
differentiation.
• It has been hypothesized that these transporter proteins are
important for maintaining the long-term proliferative
potential of stem cells.
• And lastly it will be interesting to investigate if MDR
transporter proteins are also segregated asymmetrically
during stem cell divisions and have any role in ageing and
senescence in multi-cellular organisms.
Thank you