3. RESULTS
3.2 E XPANSION OF SUBSTRATE SPECTRUM OF THE TIM22 COMPLEX
3.2.3 Import analysis of identified substrates
3.2.3.1 TIM23 complex substrate import is not affected in tim22-14 mitochondria
Figure 32 Import of TIM23 complex substrates is not affected in tim22-14 mitochondria- [35S] labelled A. F1b and B. Atp5 was imported for the indicated time points into WT and tim22-14 mitochondria. The reaction was stopped by addition of AVO. Samples were analysed by SDS-PAGE and autoradiography. p - precursor, m- mature form. AVO - mixture of 10 mM Antimycin A, 1 mM Valinomycin and 10 mM Oligomycin.
For several putative carrier substrates selected from the screen, it was decided to test their import and assembly into mitochondria to verify their dependence on TIM22. To rule out any indirect effects of the Tim22 mutant on substrates of the TIM23 complex, which utilise a presequence, import of Su9-DHFR and Atp5 was carried out. 35S labelled N-terminal part of the complex V subunit 9 fused to DHFR was synthesised
(
Figure 32A,
lane 1
).
When this precursor was incubated with mitochondria isolated from WT and tim22-14 for increasing time points, a faster migrating processed mature (m) form was generated, demonstrating efficient import and processing. Time-dependent kinetic increase could be observed, and both mitochondria were equally competent for import of a presequence-containing protein(
Figure 32A, lane 2,3,4 vs lane 6,7,8).
No mature form could be detected in the absence of a membrane potential(
Figure 32A,
lane 5 and lanelysate 10 15 15
Results
9
).
Similarly, for 35S labelled Atp5 of complex V, another TIM23 complex substrate, import efficiency into WT and tim22-14mitochondria was similar(
Figure 32B).
This Tim22 mutant strain has previously been reported to have only slightly reduced membrane potential compared to WT (Wagner et al., 2008). Therefore, any import defect due to a loss of membrane potential can be excluded.
3.2.3.2 Assembly of carrier proteins is affected in tim22-14 mitochondria
To monitor the import and assembly of selected carrier proteins from the MS analysis, blue native electrophoresis was used to differentiate between the stage III assembled and fully assembled protein. Proteins were divided into three sub-groups: (i) Carrier proteins with standard six transmembrane topology (Crc1, Odc1, Mir1, Hem25) (ii) Uncharacterised proteins potentially belonging to the carrier family of proteins (YFR045W, YPR011C) and (iii) Proteins with unusual number of transmembrane domains for carrier substrates (MPC1, MPC3). MPC1 and MPC3 have 2 and 3 predicted transmembrane domains each.For proteins from (i) and (ii), 35S labelled precursors were imported and assembled into heat-shocked WT and tim22-14 mitochondria. Crc1 (Carnitine carrier) assembly was strongly reduced in tim22-14 mitochondria compared to WT mitochondria
(
Figure 33A,
lane 1,2,3 vs lane 5,6,7
).
The complex did not assemble in the ∆Y control(
Figure 33A,
lane 4 and 8
).
For Odc1 (OxoDicarboxylate carrier), the decrease in import and assembly was not as strong, but still evident(
Figure 33B, lane 1,2,3 vs lane 5,6,7).
Assembly of glycine transporter Hem25 (HEMe synthesis by SLC25 family member) was strongly inhibited in the Tim22 mutant (Figure 33C, lane 1,2,3 vs lane 5,6,7). Mir1 or Phosphate carrier (PiC) subunit assembly was also reduced, but not to a great extent (Figure 33D, lane 1,2,3 vs lane 5,6,7). Taken together, these results show that carrier proteins identified from the quantitative MS analysis are indeed substrates of the TIM22 complex. These proteins are affected to varying degrees in their assembly, which could be due to differences in the affinity of their internal signals for the TIM22 complex.One of the uncharacterised proteins identified in the screen, YFR045W, also has six predicted transmembrane segments and is a putative mitochondrial transporter protein (Belenkiy et al., 2000). Import and assembly of 35S YFRO45W was carried out as described before. Strong assembly defects were observed (Figure 34, lane 1,2,3 vs lane 5,6,7), leading to the conclusion that YFRO45W is also a TIM22 complex substrate.
Figure 33 Assembly of carrier proteins is affected in tim22-14 mitochondria - [35S]
labelled A. Crc1, B. Odc1, C. Hem25 and 4. Mir1 was imported for 15, 30 and 60 min into WT and tim22-14 mitochondria to assess the assembly of carrier proteins. The reaction was stopped by addition of AVO followed by PK digestion. Assembly of the carrier was monitored on BN-PAGE followed by autoradiography. -*: non-specific signal. AVO - mixture of 10 mM Antimycin A, 1 mM Valinomycin and 10 mM Oligomycin.
YPR011C is characterised to be a transporter of adenosine 5’-phosphosulfate in yeast (Todisco et al., 2014). 35S YPR011C was imported and assembled into WT and tim22-14.
Assembly was reduced in the Tim22 mutant, indicating that YPR011C is also dependent on the TIM22 complex for its import (Figure 35, lane 1,2,3 vs lane 5,6,7).
Results
Figure 34 Assembly of uncharacterised carrier protein YFR045W is affected in tim22-14 mitochondria - [35S] labelled YFR045W was imported for 15, 30 and 60 min into WT and tim22-14 mitochondria to assess its assembly. The reaction was stopped by addition of AVO followed by PK digestion. Assembly was monitored on BN-PAGE followed by autoradiography. -*: non-specific signal. AVO - mixture of 10 mM Antimycin A, 1 mM Valinomycin and 10 mM Oligomycin.
Figure 35 Assembly of uncharacterised protein YPR011C is reduced in tim22-14 mitochondria - [35S] labelled YPR011C was imported for 15, 30 and 60 min into WT and tim22-14 mitochondria to assess its assembly. The reaction was stopped by addition of AVO followed by PK digestion. Assembly was monitored on BN-PAGE followed by autoradiography. -*: non-specific signal. AVO - mixture of 10 mM Antimycin A, 1 mM Valinomycin and 10 mM Oligomycin.
30 60 60
Taken together, these results confirm some of the established carrier family proteins such as phosphate carrier (PiC), carnitine carrier (Crc1), glycine transporter (Hem25) and 2-oxodicarboxylate carrier (Odc1) as substrates of the TIM22 complex. Additionally, uncharacterised proteins YFR045W and YPR011C, which have previously been reported to belong to the carrier family of proteins by proteomic studies, were confirmed as substrates of the TIM22 complex. Therefore, this quantitative mass spectrometry based approach helped in unravelling a set of inner membrane proteins which depend on TIM22 for their import.