TITLE: Reconstitution of human PINK1 and outer mitochondria TOM complex in yeast
AUTHORS: Olawale G. Raimi, Miratul Muqit

[DESCRIPTION]
PTEN induced kinase 1(PINK1) is a mitochondria kinase that phosphorylates ubiquitin and Ubl domain of parkin coincidentally at structurally obscured S65 in both proteins and initiate mitophagy. Dysregulation of this process has been implicated in cancer, obesity, cardiac disease, and neurodegenerative diseases (Youle and Narendra, 2011). PINK1 and TOM complex are key regulators in the mitochondrial quality control pathway. Physiologically under ideal condition PINK1 via its MTS forms a complex with the tom complex comprising of seven individual toms (TOMs 5, 6, 7, 20, 22, 40 and 70) going through the pores of TOM40 into the inner mitochondria (IMM) (Rasool et al., 2022). In the IMM PINK1 is cleaved within the MTS region precisely between residues 103 and 104 by two IMM proteases, MPP and PARL. The processed PINK1 is then released back into the cytosol where it is channelled towards proteasomal degradation (Rasool et al., 2022). In a pathological condition however, that’s upon mitochondrial damage, PINK1 import is blocked, preventing its processing. This results in PINK1 accumulation at the outer mitochondria membrane (OMM) and the activation of PINK1 through autophosphorylation, which subsequently initiates the mitophagy pathway to prevent the accumulation of reactive oxygen species from damaged mitochondria which could lead to cell apoptosis (Rasool et al., 2022). Here we describe in detail the protocol for making yeast cells stably expressing PINK1 and Human TOMs which has allowed us to characterise PINK1 when co-express with the TOMs. 

[STEPS]
SECTION: Miniprep
1. Before cloning, codon optimise the human genes for yeast expression. To scale up the plasmids, carry out miniprep using the Qiagen miniprep kit following the manufacturer’s instructions.


 
 
 

SECTION: Transformation of yeast cells
2. 
 

To start with the first plasmid, make   culture of MATa cells in 5 mL of YP medium supplemented with 2% glucose. Grow at 30 °C with shaking at 180 rpm.

SECTION: Transformation of yeast cells
3. The following morning prepare a 50 mL YP medium containing 2% glucose and inoculate with 5 % overnight culture. Grow at 30 °C with shaking at 180 rpm. 

SECTION: Transformation of yeast cells
4. While waiting for the right cell density, linearise DNA using the appropriate restriction enzyme for 120 min at 30 °C with shaking using a thermomixer.

SECTION: Transformation of yeast cells
5. To do this you need 4 µL of the enzyme buffer in a sterile Eppendorf tube, followed by 2 µL of the restriction enzyme (for pOR1 its Pml1, pOR3 its Nhe1, pOR2 its Ndel1 and pOR4 its Afl11), then 2 µg of DNA and make up to 40 µL with sterile water and allowed to digest for 120 min.
 


SECTION: Transformation of yeast cells
6. At an optical density OD600 of ~1.6 or a cell count of 2 x 107 cells/ml collect cells by centrifugation using tabletop Eppendorf centrifuge (model 5810R) at 4000 rpm for 10 min.

SECTION: Transformation of yeast cells
7. Decant supernatant and wash cells with sterile water twice each time collect cells by centrifugation.
SECTION: Transformation of yeast cells
8. After the last wash resuspend cells with 900 mL of sterile water and from this take 200 µL into a sterile Eppendorf tube and collect cells by centrifugation using a small tabletop centrifuge at highest speed for 5 min.

SECTION: Transformation of yeast cells
9. Remove supernatant and keep on ice.

SECTION: Transformation of yeast cells
10. To transform the cells with the DNA, in a sterile environment using flame, add 240 µL of sterile Polyetheleneglycol, 36 µL of sterile lithium acetate followed by 50 µL of 10% charged SS DNA and lastly the linearised DNA. 

SECTION: Transformation of yeast cells
11. Vortex and incubate at 42 °C for 20 min.

SECTION: Transformation of yeast cells
12. After the heat shock, collect cells by centrifugation and resuspend with 200 µL of sterile water and plate on a synthetic complete (SC) agar plate with an appropriate dropout supplement. For pOR1 its -TRP, pOR2 its -URA3, pOR3 its -HIS and pOR4 its -LEU. 

SECTION: Transformation of yeast cells
13. Cells can be split into 30-70% on two plates and a third negative control plate with the  untransformed cells. 
SECTION: Transformation of yeast cells
14. Incubate plates at 30 °C for 2880 min. 

SECTION: Screening for positive clone
15. After 2880 min colonies should have appeared with the 70% plate containing more dense colonies compared with less dense and well disperse colonies on the 30% plates.

SECTION: Screening for positive clone
16. Select four colonies and do a test expression for the desired protein. 
SECTION: Screening for positive clone
17. Streak the four colonies separately on a yeast peptone (YP) agar plate divided into four equal partitions and incubate at 30 °C 2880 min. 

SECTION: Making overnight culture
18. Make overnight cultures of the four colonies differently by inoculating 5 mL YP medium supplemented with 2% raffinose with a tiny scoop of each clone and grow at 30 °C with shaking at 180 rpm in an Infors shaker designated for yeast. 

SECTION: Protein expression
19. Make a 10 mL culture using YP medium supplemented with 2% raffinose and inoculate with 5% of the overnight culture and grow at 30 °C with shaking as for the overnight culture. 

SECTION: Protein expression
20. At the right optical density (OD600 of ~1.7) or cells count of 3 x 107 cells/ml, induce protein expression by adding to a final concentration 2% sterile galactose under a sterile condition (close to a flame).
SECTION: Protein expression
21. Continue growing for a further 10-12 h usually 2880 min. 

SECTION: Harvesting of cells
22. Harvest cell by centrifugation at 4000 rpm for 10 min using a tabletop Eppendorf centrifuge.

SECTION: Harvesting of cells
23. Decant supernatant and place cells on ice. 

SECTION: Protein extraction
24. Perform protein extraction using TCA protein extraction and lyse cells by vortexing in the presence of small amount of glass beads in an Eppendorf tube. 
SECTION: Protein extraction
25. First resuspend cells in 200 µL of 20% TCA and transfer into 1.5 ml Eppendorf tube.

SECTION: Protein extraction
26. Add small amount of glass beads and vortex for 35 s.

SECTION: Protein extraction
27. Transfer 50 µL of the lysed cells into a clean and well-labelled tube.

SECTION: Protein extraction
28. Add another 200 µL of 5% TCA, vortex again for 35 s.

SECTION: Protein extraction
29. Take 150 µL and add to the initial 50 µL.

SECTION: Protein extraction
30. Collect precipitated protein by centrifugation at high-speed using refrigerated tabletop centrifuge.
SECTION: Protein extraction
31. Decant supernatant and resuspend protein in a 200 µL lysis buffer. 

SECTION: Protein quantification
32. Quantify protein using BCA. To do this make 1:10 dilution of the cell lysate.
SECTION: Protein quantification
33. Mix 10 µL of these samples along with protein standard (Albumin) with 200 µL of BCA reagent.

SECTION: Protein quantification
34. Incubate at 37 °C for 25 min.

SECTION: Protein quantification
35. Read absorbance at 562 nm and extrapolate concentration using the protein standard curve. 
SECTION: Western blot
36. Perform western blot to check for the expression of the desired proteins, PINK1 activity by blotting for phospho-ubiquitin or other housekeeping genes. To do this, run 20 µg of protein on SDS-PAGE and transfer protein on a nitrocellulose membrane.

SECTION: Western blot
37. After the run, block in 5% milk in TBST for 60 min.

SECTION: Western blot
38. Wash membrane in TBST (3 x 10 minutes).
SECTION: Western blot
38.1. Wash membrane in TBST (3 x 10 min) (1/3).
SECTION: Western blot
38.2. Wash membrane in TBST (3 x 10 min) (2/3).
SECTION: Western blot
38.3. Wash membrane in TBST (3 x 10 min) (3/3).
SECTION: Western blot
39. Incubate in primary antibody 10 min at 4 °C.

SECTION: Western blot
40. Next day wash membrane as before with TBST and incubate membrane in secondary antibody at Room temperature for 60 min.

SECTION: Western blot
41. Wash membrane again in TBST and scan membrane on the LiCor machine. 

 

SECTION: Western blot
42. Transform these cells with the second plasmid as described above from Transformation of yeast cells section to the Western blot section. 
SECTION: Western blot
43. Repeat these steps until you have obtained cells with all the four plasmids and capable of expressing all the eight proteins. 
SECTION: Western blot
44. Also make a glycerol stock of a positive clone expressing all the eight proteins.