Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Oct 26;96(22):12400-5.
doi: 10.1073/pnas.96.22.12400.

Isolation of a Chinese hamster ovary cell mutant defective in intramitochondrial transport of phosphatidylserine

K Emoto  1 , O Kuge, M Nishijima, M Umeda
Affiliations

Isolation of a Chinese hamster ovary cell mutant defective in intramitochondrial transport of phosphatidylserine

K Emoto et al. Proc Natl Acad Sci U S A. .

Abstract

A CHO-K1 cell mutant with a specific decrease in cellular phosphatidylethanolamine (PE) level was isolated as a variant resistant to Ro09-0198, a PE-directed antibiotic peptide. The mutant was defective in the phosphatidylserine (PS) decarboxylation pathway for PE formation, in which PS produced in the endoplasmic reticulum is transported to mitochondria and then decarboxylated by an inner mitochondrial membrane enzyme, PS decarboxylase. Neither PS formation nor PS decarboxylase activity was reduced in the mutant, implying that the mutant is defective in some step of PS transport. The transport processes of phospholipids between the outer and inner mitochondrial membrane were analyzed by use of isolated mitochondria and two fluorescence-labeled phospholipid analogs, 1-palmitoyl-2-[N-[6(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-PS (C6-NBD-PS) and C6-NBD-phosphatidylcholine (C6-NBD-PC). On incubation with the CHO-K1 mitochondria, C6-NBD-PS was readily decarboxylated to C6-NBD-PE, suggesting that the PS analog was partitioned into the outer leaflet of mitochondria and then translocated to the inner mitochondrial membrane. The rate of decarboxylation of C6-NBD-PS in the mutant mitochondria was reduced to approximately 40% of that in the CHO-K1 mitochondria. The quantity of phospholipid analogs translocated from the outer leaflet of mitochondria into inner mitochondrial membranes was further examined by selective extraction of the analogs from the outer leaflet of mitochondria. In the mutant mitochondria, the translocation of C6-NBD-PS was significantly reduced, whereas the translocation of C6-NBD-PC was not affected. These results indicate that the mutant is defective in PS transport between the outer and inner mitochondrial membrane and provide genetic evidence for the existence of a specific mechanism for intramitochondrial transport of PS.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Cytolytic sensitivity of CHO-K1 and R-41 mutant cells to Ro. Cells were seeded in a 96-well culture plate at 1 × 104 cells per well. After incubation for 2 days at 39.5°C, the cells were washed twice with F-12 medium and then incubated with various concentrations of Ro peptide for 1 h at 39.5°C. Cell viability was determined by measuring the activity of lactate dehydrogenase released from dead cells. Values are means from three experiments. ○, CHO-K1; ●, R-41.
Figure 2
Figure 2
R-41 mutant is defective in the PS decarboxylation pathway for PE formation. The time course of [14C]serine incorporation into PE (A) and PS (B) in CHO-K1 and R-41 mutant cells is shown. (C) The time course of [14C]ethanolamine incorporation into PE in CHO-K1 and R-41 mutant cells is shown. Cells were seeded in 60-mm diameter dishes and cultured to a density of ≈5 × 105 cells per dish in growth medium at 39.5°C. The cells were metabolically labeled at 39.5°C by replacing the medium with 2 ml of fresh growth medium containing l-[U-14C]serine (0.5 μCi/ml) or [1,2-14C]ethanolamine (0.5 μCi/ml). At the indicated times, cellular phospholipids were extracted from one dish of each strain and analyzed by TLC. The number of cells at zero time was determined and used to standardize the results. (D) Turnover of PE pulse-labeled with [14C]serine in CHO-K1 and R-41 mutant cells is shown. Cells were incubated with l-[U-14C]serine (2 μCi/ml) for 1 h at 39.5°C. After removing the radioactive medium (zero time), cells were washed and further incubated in chase medium containing 1 mM serine and 1 mM ethanolamine (29). At the indicated times, the cells were harvested, and phospholipids were extracted and analyzed by one-dimensional TLC. ○, CHO-K1; ●, R-41.
Figure 3
Figure 3
PSD activity in CHO-K1 and R-41 mutant cells. (A) Cells seeded at 5 × 105 cells per 100-mm dish were grown at 39.5°C for 3 days. The harvested cells were sonically disrupted in 0.25 M sucrose, 10 mM Hepes (pH 7.5), and 1 mM EDTA, and the cell lysate were assayed for PSD activity as described (25). The error for duplicate determinations was less than 5%. ○, CHO-K1; ●, R-41. (B) Effect of expression of a CHO PSD cDNA on PE formation via the PS decarboxylation pathway. CHO-K1 (○, ▵) and R-41 mutant (●, ▴) cells transfected either with control vector (mock; ○, ●) or with vector containing cDNA of PSD gene (PSD; ▵, ▴) were cultured for 2 days at 39.5°C. The cells were metabolically labeled with l-[U-14C]serine (0.5 μCi/ml). At the indicated times, cellular phospholipids were extracted from one dish of each strain and analyzed by TLC.
Figure 4
Figure 4
Posttranslational processing of the PSD gene product. CHO-K1 and R-41 mutant cells transfected with the PSD gene were pulse-labeled with [35S]methionine for 5 min and then chased in the presence of excess unlabeled methionine for the periods indicated. Radioactive proteins were precipitated with anti-PSD antibody, separated by SDS/PAGE on 12% gels, and examined by use of a bioimage analyzer.
Figure 5
Figure 5
Translocation-dependent decarboxylation of C6-NBD-PS in isolated mitochondria from CHO-K1 and R-41 mutant cells. Intact (A) or disrupted (B) mitochondria from either CHO-K1 or R-41 mutant cells were incubated with 10 μM C6-NBD-PS in 1 ml of buffer A (250 mM mannitol/5 mM Hepes, pH 7.4/0.5 mM EGTA) at 39.5°C for various periods. The reaction was terminated by adding 3.75 ml of chloroform/methanol, 1:2 (vol/vol), and then the lipids were extracted and separated by TLC. C6-NBD-PE spots were scraped from TLC plates, and then the lipids were extracted as described by Bligh and Dyer (26). The relative fluorescence of the lipid extracts was measured with a fluorescence spectrophotometer F-2000 (Hitachi, Tokyo). Mitochondria were disrupted by exposure five times to ultrasound on ice for 30 sec at setting 1 in a Branson ultrasonic disrupter. ○, CHO-K1; ●, R-41. R.F.U., relative fluorescent unit(s).
Figure 6
Figure 6
Association of C6-NBD-lipids with isolated mitochondria from CHO-K1 and R-41 mutant cells. Mitochondria from CHO-K1 or R-41 mutant cells were incubated with 10 μM C6-NBD-PS (A and B) or C6-NBD-PC (C and D) at 30°C, and then total C6-NBD-lipids associated with mitochondria were extracted and quantified (A and C). (B and D) Mitochondria incubated with C6-NBD-lipids were subsequently subject to back exchange to remove the fluorescent lipids from the outer leaflet of the outer mitochondrial membrane, and then C6-NBD-lipids remaining in mitochondria were extracted and quantified. The amount of C6-NBD-lipids was normalized to total mitochondrial proteins. ○, CHO-K1; ●, R-41.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Schatz G. J Biol Chem. 1996;271:31763–31766. - PubMed
    1. Neupert W. Annu Rev Biochem. 1997;66:863–917. - PubMed
    1. Daum G. Biochim Biophys Acta. 1985;822:1–42. - PubMed
    1. Vance J E. J Biol Chem. 1991;266:89–97. - PubMed
    1. Dennis E A, Kennedy E P. J Lipid Res. 1972;13:263–267. - PubMed

Substances

LinkOut - more resources