Pyrroline-5-carboxylate synthase (P5CS) catalyzes the synthesis of pyrroline-5-carboxylate (P5C), a key precursor for the synthesis of proline and ornithine. P5CS malfunction leads to multiple human diseases; however, the molecular mechanism underlying these diseases is unknown. We found that P5CS localizes in mitochondria in rod- and ring-like patterns but diffuses inside the mitochondria upon cellular starvation or exposure to oxidizing agents. Some of the human disease-related mutant forms of P5CS also exhibit diffused distribution. Multimerization (but not the catalytic activity) of P5CS regulates its localization. P5CS mutant cells have a reduced proliferation rate and are sensitive to cellular stresses. Flies lacking P5CS have reduced eclosion rates. Lipid droplets accumulate in the eyes of the newly eclosed P5CS mutant flies, which degenerate with aging. The loss of P5CS in cells leads to abnormal purine metabolism and lipid-droplet accumulation. The reduced lipid-droplet consumption is likely due to decreased expression of the fatty acid transporter, CPT1, and few β-oxidation-related genes following P5CS knockdown. Surprisingly, we found that P5CS is required for mitochondrial respiratory complex organization and that the respiration defects in P5CS knockout cells likely contribute to the metabolic defects in purine synthesis and lipid consumption. This study links amino acid synthesis with mitochondrial respiration and other key metabolic processes, whose imbalance might contribute to P5CS-related disease conditions.
Subscribe to Journal
Get full journal access for 1 year
only $41.58 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Martinez-Reyes I, Chandel NS. Mitochondrial TCA cycle metabolites control physiology and disease. Nat Commun. 2020;11:102.
Spinelli JB, Haigis MC. The multifaceted contributions of mitochondria to cellular metabolism. Nat cell Biol. 2018;20:745–54.
Kauppila TES, Kauppila JHK, Larsson NG. Mammalian mitochondria and aging: an update. Cell Metab. 2017;25:57–71.
Sun N, Youle RJ, Finkel T. The mitochondrial basis of aging. Mol Cell. 2016;61:654–66.
Perez-Arellano I, Carmona-Alvarez F, Martinez AI, Rodriguez-Diaz J, Cervera J. Pyrroline-5-carboxylate synthase and proline biosynthesis: from osmotolerance to rare metabolic disease. Protein Sci: Publ Protein Soc. 2010;19:372–82.
Panza E, Escamilla-Honrubia JM, Marco-Marin C, Gougeard N, De Michele G, Morra VB, et al. ALDH18A1 gene mutations cause dominant spastic paraplegia SPG9: loss of function effect and plausibility of a dominant negative mechanism. Brain: J Neurol. 2016;139:e3.
Koh K, Ishiura H, Beppu M, Shimazaki H, Ichinose Y, Mitsui J, et al. Novel mutations in the ALDH18A1 gene in complicated hereditary spastic paraplegia with cerebellar ataxia and cognitive impairment. J Hum Genet. 2018;63:1009–13.
Skidmore DL, Chitayat D, Morgan T, Hinek A, Fischer B, Dimopoulou A, et al. Further expansion of the phenotypic spectrum associated with mutations in ALDH18A1, encoding Delta(1)-pyrroline-5-carboxylate synthase (P5CS). Am J Med Genet Part A. 2011;155A:1848–56.
Wolthuis DF, van Asbeck E, Mohamed M, Gardeitchik T, Lim-Melia ER, Wevers RA, et al. Cutis laxa, fat pads and retinopathy due to ALDH18A1 mutation and review of the literature. Eur J Paediatr Neurol: Off J Eur Paediatr Neurol Soc. 2014;18:511–5.
Fischer-Zirnsak B, Escande-Beillard N, Ganesh J, Tan YX, Al Bughaili M, Lin AE, et al. Recurrent de novo mutations affecting residue Arg138 of pyrroline-5-carboxylate synthase cause a progeroid form of autosomal-dominant cutis laxa. Am J Hum Genet. 2015;97:483–92.
Krishnan N, Dickman MB, Becker DF. Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress. Free Radic Biol Med. 2008;44:671–81.
Shafqat S, Velaz-Faircloth M, Henzi VA, Whitney KD, Yang-Feng TL, Seldin MF, et al. Human brain-specific L-proline transporter: molecular cloning, functional expression, and chromosomal localization of the gene in human and mouse genomes. Mol Pharmacol. 1995;48:219–29.
Wang D, Liu Y, Zhang R, Zhang F, Sui W, Chen L, et al. Apoptotic transition of senescent cells accompanied with mitochondrial hyper-function. Oncotarget. 2016;7:28286–300.
Rebbaa A, Zheng X, Chou PM, Mirkin BL. Caspase inhibition switches doxorubicin-induced apoptosis to senescence. Oncogene. 2003;22:2805–11.
Perez-Arellano I, Rubio V, Cervera J. Mapping active site residues in glutamate-5-kinase. The substrate glutamate and the feed-back inhibitor proline bind at overlapping sites. FEBS Lett. 2006;580:6247–53.
Marco-Marin C, Gil-Ortiz F, Perez-Arellano I, Cervera J, Fita I, Rubio V. A novel two-domain architecture within the amino acid kinase enzyme family revealed by the crystal structure of Escherichia coli glutamate 5-kinase. J Mol Biol. 2007;367:1431–46.
Page R, Nelson MS, von Delft F, Elsliger MA, Canaves JM, Brinen LS, et al. Crystal structure of gamma-glutamyl phosphate reductase (TM0293) from Thermotoga maritima at 2.0 A resolution. Proteins. 2004;54:157–61.
Liu ZJ, Sun YJ, Rose J, Chung YJ, Hsiao CD, Chang WR, et al. The first structure of an aldehyde dehydrogenase reveals novel interactions between NAD and the Rossmann fold. Nat Struct Biol. 1997;4:317–26.
Wu CA, Chao Y, Shiah SG, Lin WW. Nutrient deprivation induces the Warburg effect through ROS/AMPK-dependent activation of pyruvate dehydrogenase kinase. Biochimica Biophysica Acta. 2013;1833:1147–56.
Meister A, Anderson ME. Glutathione. Annu Rev Biochem. 1983;52:711–60.
Rambold AS, Cohen S, Lippincott-Schwartz J. Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics. Developmental Cell. 2015;32:678–92.
Liu L, Zhang K, Sandoval H, Yamamoto S, Jaiswal M, Sanz E, et al. Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration. Cell. 2015;160:177–90.
Harris CA, Haas JT, Streeper RS, Stone SJ, Kumari M, Yang K, et al. DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes. J lipid Res. 2011;52:657–67.
Nguyen TB, Louie SM, Daniele JR, Tran Q, Dillin A, Zoncu R, et al. DGAT1-dependent lipid droplet biogenesis protects mitochondrial function during starvation-induced autophagy. Developmental Cell. 2017;42:9–21 e25.
Mayer N, Schweiger M, Romauch M, Grabner GF, Eichmann TO, Fuchs E, et al. Development of small-molecule inhibitors targeting adipose triglyceride lipase. Nat Chem Biol. 2013;9:785–7.
Cuthbert KD, Dyck JR. Malonyl-CoA decarboxylase is a major regulator of myocardial fatty acid oxidation. Curr hypertension Rep. 2005;7:407–11.
Konovalova S. Analysis of mitochondrial respiratory chain complexes in cultured human cells using blue native polyacrylamide gel electrophoresis and immunoblotting. J Vis Exp. 2019;144. https://doi.org/10.3791/59269.
French JB, Jones SA, Deng H, Pedley AM, Kim D, Chan CY, et al. Spatial colocalization and functional link of purinosomes with mitochondria. Science. 2016;351:733–7.
Pedley AM, Benkovic SJ. A new view into the regulation of purine metabolism: the purinosome. Trends Biochem Sci. 2017;42:141–54.
Vanakker O, Callewaert B, Malfait F, Coucke P. The genetics of soft connective tissue disorders. Annu Rev Genom Hum Genet. 2015;16:229–55.
Ignatova Z, Gierasch LM. Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant. Proc Natl Acad Sci USA. 2006;103:13357–61.
Ren X, Sun J, Housden BE, Hu Y, Roesel C, Lin S, et al. Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9. Proc Natl Acad Sci USA. 2013;110:19012–7.
Zhang Y, Liu X, Bai J, Tian X, Zhao X, Liu W, et al. Mitoguardin regulates mitochondrial fusion through MitoPLD and is required for neuronal homeostasis. Mol Cell. 2016;61:111–24.
Ariotti N, Hall TE, Rae J, Ferguson C, McMahon KA, Martel N, et al. Modular detection of GFP-labeled proteins for rapid screening by electron microscopy in cells and organisms. Developmental Cell. 2015;35:513–25.
Sellick CA, Hansen R, Stephens GM, Goodacre R, Dickson AJ. Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling. Nat Protoc. 2011;6:1241–9.
Xiao Z, Cheng G, Jiao Y, Pan C, Li R, Jia D, et al. Holo-Seq: single-cell sequencing of holo-transcriptome. Genome Biol. 2018;19:163.
We are grateful to THFC, BDSC, and DGRC for providing fly strains and cDNA clones. We thank the imaging core facilities and mass spectrometry facilities in LSI for the technical support. CT is supported by National Natural Science Foundation of China (91754103, 31622034, 31571383), National Key Research & Developmental Program of China (2017YFC1001500, 2017YFC1001100), Natural Science Foundation of Zhejiang Province, China (LR16C070001) and Fundamental research funds for the central universities. CT is a Qianjiang Scholar.
Conflict of interest
The authors declare that they have no conflict of interest.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Edited by L. Scorrano
About this article
Cite this article
Yang, Z., Zhao, X., Shang, W. et al. Pyrroline-5-carboxylate synthase senses cellular stress and modulates metabolism by regulating mitochondrial respiration. Cell Death Differ 28, 303–319 (2021). https://doi.org/10.1038/s41418-020-0601-5