Iron-regulated assembly of the cytosolic iron–sulfur cluster biogenesis machinery

Abstract

The cytosolic iron–sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron–sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization. The cytosolic iron–sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron–sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization. Iron–sulfur (Fe-S) clusters are ubiquitous cofactors utilized by all realms of life, among which [2Fe-2S] and [4Fe-4S] clusters are the most commonly found in biological systems (1Crack J.C. Green J. Thomson A.J. Le Brun N.E. Iron-sulfur cluster sensor-regulators.Curr. Opin. Chem. Biol. 2012; 16: 35-44Crossref PubMed Scopus (84) Google Scholar). These cofactors play a role in maintaining protein stability, as well as regulating subcellular localization and enzymatic activity (2Stehling O. Vashisht A.A. Mascarenhas J. Jonsson Z.O. Sharma T. Netz D.J. et al.MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity.Science. 2012; 337: 195-199Crossref PubMed Scopus (226) Google Scholar, 3Ben-Shimon L. Paul V.D. David-Kadoch G. Volpe M. Stümpfig M. Bill E. et al.Fe-S cluster coordination of the chromokinesin KIF4A alters its sub-cellular localization during mitosis.J. Cell Sci. 2018; 131: jcs211433Crossref PubMed Google Scholar, 4Beinert H. Holm R.H. Munck E. Iron-sulfur clusters: nature’s modular, multipurpose structures.Science. 1997; 277: 653-659Crossref PubMed Scopus (1547) Google Scholar). Being redox sensitive, these clusters also serve as redox centers to facilitate electron transfer. The redox states of Fe-S clusters change in response to environmental stimuli, which provides an additional layer of regulation of protein function (1Crack J.C. Green J. Thomson A.J. Le Brun N.E. Iron-sulfur cluster sensor-regulators.Curr. Opin. Chem. Biol. 2012; 16: 35-44Crossref PubMed Scopus (84) Google Scholar, 4Beinert H. Holm R.H. Munck E. Iron-sulfur clusters: nature’s modular, multipurpose structures.Science. 1997; 277: 653-659Crossref PubMed Scopus (1547) Google Scholar). In eukaryotic organisms, the biogenesis of Fe-S clusters is highly compartmentalized with distinct branches of the biogenesis pathway responsible for the maturation of mitochondrial and extramitochondrial Fe-S proteins (5Maio N. Rouault T.A. Outlining the complex pathway of mammalian Fe-S cluster biogenesis.Trends Biochem. Sci. 2020; 45: 411-426Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 6Lill R. Srinivasan V. Mühlenhoff U. The role of mitochondria in cytosolic-nuclear iron-sulfur protein biogenesis and in cellular iron regulation.Curr. Opin. Microbiol. 2014; 22: 111-119Crossref PubMed Scopus (104) Google Scholar). The maturation of extramitochondrial Fe-S proteins is facilitated specifically by the cytosolic Fe-S cluster biogenesis pathway (CIA). The CIA pathway is associated with a plethora of cellular processes, including cell proliferation, DNA damage repair, nonsense-mediated decay, apoptosis, and microtubule-based processes such as ciliogenesis (2Stehling O. Vashisht A.A. Mascarenhas J. Jonsson Z.O. Sharma T. 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Lysandrou C. et al.The nucleotide-binding proteins Nubp1 and Nubp2 are negative regulators of ciliogenesis.Cell Mol. Life Sci. 2014; 71: 517-538Crossref PubMed Scopus (21) Google Scholar, 11Stehling O. Netz D.J. Niggemeyer B. Rösser R. Eisenstein R.S. Puccio H. et al.Human Nbp35 is essential for both cytosolic iron-sulfur protein assembly and iron homeostasis.Mol. Cell Biol. 2008; 28: 5517-5528Crossref PubMed Scopus (94) Google Scholar). Deregulation of CIA components and substrates has also been linked to numerous human diseases (5Maio N. Rouault T.A. Outlining the complex pathway of mammalian Fe-S cluster biogenesis.Trends Biochem. Sci. 2020; 45: 411-426Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 12Sheftel A. Stehling O. Lill R. Iron-sulfur proteins in health and disease.Trends Endocrinol. Metab. 2010; 21: 302-314Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 13Fuss J.O. Tsai C.L. Ishida J.P. Tainer J.A. Emerging critical roles of Fe-S clusters in DNA replication and repair.Biochim. Biophys. Acta. 2015; 1853: 1253-1271Crossref PubMed Scopus (177) Google Scholar). The maturation of cytosolic Fe-S proteins is a multistep process that is tightly regulated. In human cells, bioavailable iron is delivered for [2Fe-2S] cluster biogenesis by poly(rC)-binding protein 1 (PCBP1) to the chaperone consisting of the BolA-like protein 2 (BOLA2) and glutaredoxin-3 (GLRX3) (14Patel S.J. Frey A.G. Palenchar D.J. Achar S. Bullough K.Z. Vashisht A. et al.A PCBP1–BolA2 chaperone complex delivers iron for cytosolic [2Fe–2S] cluster assembly.Nat. Chem. Biol. 2019; 15: 872-881Crossref PubMed Scopus (59) Google Scholar). [4Fe-4S] clusters are first assembled on the CIA scaffold complex composed of nucleotide-binding protein 1 (NUBP1) and nucleotide-binding protein 2 (NUBP2) (11Stehling O. Netz D.J. Niggemeyer B. Rösser R. Eisenstein R.S. Puccio H. et al.Human Nbp35 is essential for both cytosolic iron-sulfur protein assembly and iron homeostasis.Mol. Cell Biol. 2008; 28: 5517-5528Crossref PubMed Scopus (94) Google Scholar, 15Netz D.J. Pierik A.J. Stümpfig M. Mühlenhoff U. Lill R. The Cfd1-Nbp35 complex acts as a scaffold for iron-sulfur protein assembly in the yeast cytosol.Nat. Chem. Biol. 2007; 3: 278-286Crossref PubMed Scopus (152) Google Scholar). This step requires an unknown sulfur containing compound that is produced by the mitochondrial Fe-S cluster biogenesis (ISC) machinery and transported to the through the mitochondrial protein This [4Fe-4S] cluster is to the cluster protein cytosolic iron–sulfur assembly component 3 and into substrates through the activity of the CIA targeting complex composed of protein cytosolic iron-sulfur protein assembly protein cytosolic iron-sulfur assembly component (2Stehling O. Vashisht A.A. Mascarenhas J. Jonsson Z.O. Sharma T. Netz D.J. et al.MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity.Science. 2012; 337: 195-199Crossref PubMed Scopus (226) Google Scholar, K. V. H. S.J. links iron-sulfur cluster assembly to DNA 2012; 337: PubMed Scopus Google Scholar). Although the has been in numerous between Fe-S cluster biogenesis and the cellular environment such as iron and oxygen evidence is to in (1Crack J.C. Green J. Thomson A.J. Le Brun N.E. Iron-sulfur cluster sensor-regulators.Curr. Opin. Chem. Biol. 2012; 16: 35-44Crossref PubMed Scopus (84) Google Scholar, 4Beinert H. Holm R.H. Munck E. Iron-sulfur clusters: nature’s modular, multipurpose structures.Science. 1997; 277: 653-659Crossref PubMed Scopus (1547) Google Scholar). The availability of bioavailable iron to cytosolic [2Fe-2S] cluster biogenesis by the association of and A.G. Palenchar D.J. A complex as an iron-sulfur cluster chaperone for the cytosolic cluster assembly Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the maturation of extramitochondrial Fe-S proteins involved in DNA and iron homeostasis are regulated by iron and oxygen availability A.A. Sharma T. K. J.A. The association of the DNA with is regulated by the cytosolic iron-sulfur cluster assembly Biol. Chem. 2015; Full Text Full Text PDF PubMed Scopus Google Scholar, Rouault T.A. oxygen protein in PubMed Scopus Google Scholar). these the of regulation are In we a targeted proteomics assay that proteins in the CIA we to the association of the CIA targeting complex (MMS19, CIAO1, and CIAO2B) and CIA substrates and with the CIA scaffold complex component We also find that the of CIAO3 with the CIA scaffold complex is regulated by changes in cellular including changes in the iron to reactive oxygen species and changes in oxygen The of CIAO3 with the CIA targeting by these environmental is on Fe-S cluster binding by CIAO3. CIAO3 mutants that are defective in Fe-S cluster binding association with the of the CIA machinery. Together, these data suggest the of CIA metabolon composed of the CIA scaffold the CIA targeting complex and CIA The metabolon assembly is and regulated by environmental through Fe-S clusters in CIAO3. In order to how the CIA pathway to changes in cellular we by the protein of CIA components in to iron supplementation or an iron or with or the iron for and cell with CIA components as well as for protein an that iron is and protein 2 an FBXL5 that is by iron as for and A.A. X. A. et of iron homeostasis by an PubMed Scopus Google Scholar). We changes in the of CIA components or CIA substrates in Although in Fe-S cluster incorporation been to of a of Fe-S these are as a of of the Fe-S cluster assembly machinery or in proteins defective in cluster incorporation (2Stehling O. Vashisht A.A. Mascarenhas J. Jonsson Z.O. Sharma T. Netz D.J. et al.MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity.Science. 2012; 337: 195-199Crossref PubMed Scopus (226) Google Scholar, of the to the [4Fe-4S] cluster of PubMed Scopus Google Scholar). we of changes in iron on the of CIA factors and we interactions between CIA components under that components of the CIA targeting complex are in higher order complexes in to K. V. H. S.J. links iron-sulfur cluster assembly to DNA 2012; 337: PubMed Scopus Google Scholar, M. K. K. protein is an component of the human cytosolic iron-sulfur cluster assembly (CIA) machinery and in the CIA Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, N. A. Rouault T.A. iron-sulfur cluster biogenesis with the transfer to Mol. 2018; PubMed Scopus Google Scholar). of the CIA targeting complex with which in with the CIA scaffold indicating that these CIA components may into higher order complexes. we of a component of the CIA scaffold and proteins associated with NUBP2 an In to and CIAO3 which are NUBP2 we also components of the CIA targeting complex and as well as CIA substrates and in the NUBP2 and component of the targeting in to of low we a targeted proteomics assay that monitoring to the and of a of proteins to the Fe-S cluster assembly C. B. et in and for assay a Cell 2014; Full Text Full Text PDF PubMed Scopus Google Scholar). We first assay on a cell and to the and the of CIA factors CIAO1, and and CIA substrates and This targeted which provides and proteomics to NUBP2 to specifically components of the CIA scaffold complex and components of the CIA targeting complex (MMS19, CIAO1, and and CIA substrates and utilized in are in and CIAO3 as proteins and components of the CIA targeting complex (MMS19, CIAO1, and CIAO2B) also found associated with In we that CIA substrates and with NUBP2 These suggest that the CIA scaffold the CIA targeting and CIA substrates potentially into a higher order protein assembly that Fe-S cluster transfer into The assembly is that the association between the CIA scaffold complex and the CIA targeting complex the of iron availability on the assembly of CIA we utilized of CIAO3 by to the CIAO3 between and These proteomics both protein by as well as our targeted proteomics assay that specifically the of a of Fe-S machinery proteins and The proteomics that interactions between CIAO3 and proteins on the availability of including both components of the CIA scaffold complex and and In interactions between CIAO3 and the CIA targeting complex by cellular iron These by our targeted proteomics assay in which to and the of the CIA scaffold complex and the CIA targeting complex in CIAO3 and We that CIAO3 with and NUBP2 in an and that both and interactions reduced in conditions indicating that CIAO3 the CIA scaffold complex interactions between CIAO3 and the CIA targeting complex by iron and that CIAO3 also with the CIA scaffold complex in an we NUBP2 with or NUBP2 cell and with CIAO3 data that the is by the of iron and iron is through chelation We further the association between CIAO3 and its in response to in iron of We with or for 3 or We by with data that the 3 of by of chelation a in NUBP2 binding to CIAO3 as as 3 with and to as the These suggest that the response of the to changes in iron is the that regulated by iron we these interactions also by environmental we with and the on the CIAO3 and A. and in to The roles of and of and in and J. PubMed Scopus Google Scholar). to for CIAO3 cell and with both the CIA scaffold complex and the CIA targeting complex. We a CIAO3 with the CIA scaffold complex under A between CIAO3 and the CIA targeting complex also under the In to we also oxygen and its on CIAO3 We in or and its interactions by with of as a for We found that the in in these suggest that complexes are to the redox of the The of Fe-S clusters to changes in both iron availability and the redox of the cell (1Crack J.C. Green J. Thomson A.J. Le Brun N.E. Iron-sulfur cluster sensor-regulators.Curr. Opin. Chem. Biol. 2012; 16: 35-44Crossref PubMed Scopus (84) Google Scholar, J.C. et of and by the [4Fe-4S] cluster of the iron 2019; PubMed Scopus Google Scholar). we that the of these environmental changes to CIAO3 interactions on the Fe-S clusters to CIAO3 or components of we how of Fe-S cluster biogenesis the CIAO3 that cytosolic Fe-S cluster biogenesis by the CIA pathway on mitochondrial Fe-S cluster biogenesis by the pathway and that the scaffold to reduced iron incorporation and protein of both CIAO3 and CIA substrates (2Stehling O. Vashisht A.A. Mascarenhas J. Jonsson Z.O. Sharma T. Netz D.J. et al.MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity.Science. 2012; 337: 195-199Crossref PubMed Scopus (226) Google Scholar, J. Pierik A.J. Netz D.J. Mühlenhoff U. Lill R. The is essential for maturation of cytosolic and nuclear J. PubMed Scopus Google Scholar). We and of which under the of a We a reduced of CIAO3 in the with which is with (2Stehling O. Vashisht A.A. Mascarenhas J. Jonsson Z.O. Sharma T. Netz D.J. et al.MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity.Science. 2012; 337: 195-199Crossref PubMed Scopus (226) Google Scholar). of the CIAO3 complexes reduced for both the CIA scaffold complex and the CIA targeting complex. of CIAO3 interactions as in and in the of and NUBP2 with CIAO3 in response to the of In we that the interactions between CIAO3 and components of the CIA targeting complex also upon of Together, our demonstrate the assembly of CIAO3 into higher order complexes on the of a Fe-S cluster biogenesis which an essential role in and CIA has Fe-S cluster binding its and the its V. L. CIAO3 protein a complex with of the human cytosolic iron–sulfur cluster assembly Biol. Chem. 2020; PubMed Scopus Google Scholar, E. Pierik A.J. Mühlenhoff U. Lill R. role of in the assembly of iron-sulfur clusters on the CIA protein PubMed Scopus Google Scholar). that Fe-S clusters are to the cellular environment and the function of Fe-S we that CIAO3 interactions may regulated by its cluster incorporation that of CIAO3 with in the or both and in the the protein defective in binding of Fe-S clusters V. L. CIAO3 protein a complex with of the human cytosolic iron–sulfur cluster assembly Biol. Chem. 2020; PubMed Scopus Google Scholar, E. Pierik A.J. Mühlenhoff U. Lill R. role of in the assembly of iron-sulfur clusters on the CIA protein PubMed Scopus Google Scholar). on these we CIAO3 mutants with cluster incorporation and to the Fe-S cluster for CIAO3 interactions on cluster binding by CIAO3 and the of and of CIAO3 with CIA we utilized our targeted proteomics assay that CIA components and substrates as We both and CIAO3 complexes and interactions with the CIA scaffold the CIA targeting and CIA substrates to the of CIAO3 in We that interactions reduced for with our that Fe-S clusters are required for the between CIAO3 and the CIA scaffold complex The CIA targeting complex also reduced association with CIAO3 an for the a to for the and a change in the we that the association of substrates ABCE1 and with CIAO3 the CIAO3 Fe-S cluster binding which that CIAO3 may also associate with CIA substrates independently of the CIA targeting complex. we a of to that to the CIA targeting complex A.A. Sharma T. K. J.A. The association of the DNA with is regulated by the cytosolic iron-sulfur cluster assembly Biol. Chem. 2015; Full Text Full Text PDF PubMed Scopus Google Scholar). We that associated weakly with CIAO3 to suggesting that CIAO3 binding by requires the CIA targeting complex binding of and is with the that the CIA scaffold the CIA targeting and CIA substrates form higher order complexes that Fe-S protein maturation These provide evidence that Fe-S cluster incorporation into CIAO3 its interactions and its incorporation in CIA A in CIAO3 has been to associate with J. et al.A novel in nuclear a 2017; PubMed Scopus Google Scholar). of human CIAO3 on of that is an and the Fe-S cluster A and of the to PubMed Google Scholar). we that the Fe-S cluster binding to the of or cluster binding in the In the the CIAO3 mutants and with binding to the CIA scaffold complex and the CIA targeting complex binding to CIA we both and of and the for components of the CIA scaffold complex and components of the CIA targeting complex (MMS19, CIAO1, and and CIA substrates and to the interactions of with both the CIA scaffold complex and the CIA targeting complex reduced and while its association with CIA substrates of and These findings suggest that the of to into a higher order complex may to the associated with The CIA pathway Fe-S cluster incorporation into a plethora of extramitochondrial Fe-S proteins involved in a of essential cellular functions. remains a how the CIA pathway adapts to cellular to of In we a novel of regulation for cytosolic [4Fe-4S] cluster biogenesis a targeted proteomics assay to components and substrates of the CIA we demonstrate the existence of higher order CIA complexes containing the CIA scaffold the CIA targeting and CIA These higher order complexes are to environmental changes and are in response to changes in the iron oxygen tension, and data further that Fe-S cluster binding by CIAO3 is required for its interactions with the CIA scaffold complex and the CIA targeting complex. we demonstrate that the associated with to into a CIA complex the and of Although an of the components and the of the CIA pathway has to is the of the pathway and how it to cellular and environmental in the mammalian A has that the association of with in the of the cytosolic [2Fe-2S] cluster biogenesis machinery is iron and of regulation A.G. Palenchar D.J. A complex as an iron-sulfur cluster chaperone for the cytosolic cluster assembly Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). further by that the association of CIAO3 with the CIA scaffold complex is tightly to cellular iron with iron the The regulated binding of CIAO3 with the CIA scaffold complex is also by and suggesting that it is to changes in cellular These data suggest that the CIA pathway adapts to environmental cues through the of a higher order CIA complex a by which the CIA scaffold complex the of the complex. environmental changes that association with the CIA scaffold Fe-S cluster biogenesis and cluster incorporation in CIAO3 both the CIA scaffold complex and the CIA targeting complex with CIAO3. We that the Fe-S clusters in or the changes in the cellular environment and the assembly of the CIA machinery. that the Fe-S cluster of CIAO3 is while the cluster is in the of the We that these clusters play roles in regulating CIAO3 with the Fe-S cluster to changes in the cellular environment to CIAO3 interactions and the cluster required for the of the protein for environmental to its The found to associate with J. et al.A novel in nuclear a 2017; PubMed Scopus Google Scholar). on our of with the of protein and mutants with defective Fe-S cluster we a in protein for the This also weakly with the CIA scaffold complex and the CIA targeting complex. These findings are with the that the cluster of CIAO3 its and that Fe-S cluster binding in CIAO3 is required for its incorporation into CIA The in cytosolic Fe-S cluster biogenesis and potentially to the of containing human CIAO3 with the containing the CIAO3 into and into the a and with containing the by with and the The cell to and CIAO3 the in with 2 with or under conditions in with conditions by for in a with a containing and a of for the and The cell with 1 for to protein with including or of by with the and the Cell in 1 1 1 or 1 A and by for and by protein of for to the cell and for to 1 by with 1 1 or 1 and for by for in and and reduced with containing cell or by and with or with CIAO3 or NUBP2 or and FBXL5 or and further with or on or with the of with to of 2012; PubMed Scopus Google Scholar). in 1 1 1 or 1 A and reduced with with with and with in to which further to with Cell in 2 1 1 1 or 1 for by for and protein or of to the cell and for 1 by or with 1 1 or 1 and 1 or 2 additional with in by and to a of to proteins for 2 or for and the the protein by an additional of The protein and in in reduced with with and with and by of to a of with and in for by protein complexes on a A to the an by composed of with and and in A and in with A and in and and in a of the of to the to in the to in the to 1 for 2 and to the 2 A to a 3 of with to to with states between 2 and and an for by a a of or with the to data the in containing proteins with S. T. J. The for PubMed Scopus Google Scholar). The of for first and for of containing on with in including and protein for and with most as with for through a and both and protein for proteins by are in by in protein with J. N. for Scholar, M. T. T. B. et an for of 2014; PubMed Scopus Google Scholar). A to proteins with the targeted proteomics by N. a targeted and J. Mol. Sci. 2015; 16: PubMed Scopus Google Scholar). to the human to in and also to and which are to These targeted in both cell and and to as cell an of with and an to with to with the to of CIAO3 for targeted are that to to and of a first of protein complexes associated with and B. N. M. B. et an for and targeted proteomics 2010; PubMed Scopus Google Scholar). to of all for of the and to of the to its in the in the for and A of on these data and is in data for assay been to with targeted biological to of that the targeted proteins to with a of to an for and of proteomics to protein and changes conditions M. T. T. 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