SSU3 (IPMI SSU3, AtLeuD2, IPMI1; At3g58990) is one of three small subunits (LeuD family) of the plastid heterodimeric isopropylmalate isomerase (IPMI) in Arabidopsis. It pairs with the single large subunit IIL1 (LeuC), supplying the aconitase domain-4-like region that forms the substrate recognition site, and thereby determines the substrate preference of the heterodimer. Together with its close paralog SSU2, SSU3 is specialized for the methionine chain-elongation pathway of aliphatic glucosinolate biosynthesis, where the LSU1:SSU2/3 isomerase converts 2-(omega-methylthio)alkylmalates into the corresponding 3-alkylmalates in successive elongation cycles; SSU2/SSU3 are required for the formation of the longest (C7, C8) aliphatic glucosinolates. The SSU3-containing enzyme can also isomerize 2-isopropylmalate, but SSU3 contributes little to leucine biosynthesis under normal conditions, a role filled by the essential SSU1. SSU3 is co-regulated with other aliphatic glucosinolate genes by MYB28, is expressed in and around the vasculature (phloem- and xylem-associated cells) of roots, hypocotyls, leaves and inflorescence stems, and localizes to chloroplasts (stroma). Single ssu3 mutants have no visible phenotype because of redundancy with SSU2.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003861 3-isopropylmalate dehydratase activity | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT-based inference of 3-isopropylmalate dehydratase activity for the LeuD (IPMI small subunit) family. Reason: SSU3 is inside the LeuD clade and retains the ability to act on IPM: SSU3-containing IPMI has a broad substrate range including IPM (Imhof et al. 2014), and SSU2/SSU3 support residual Leu synthesis when SSU1 is knocked down. As a small subunit it contributes to the heterodimer's activity rather than possessing it alone; the activity is not its principal in vivo role (which is Met chain elongation). This retained heterodimer activity is non-core relative to SSU3 specialization in methionine chain elongation. Supporting Evidence: PMID:24608865 On the contrary, IPMI containing SSU2 or SSU3 subunits has a broad substrate range including Met derivatives with different side-chain lengths and IPM. PMID:24608865 As indicated by the normal green color a residual Leu biosynthesis occurs in or along the vasculature by the participation of IPMI SSU2 and/or IPMI SSU3. |
| GO:0003861 3-isopropylmalate dehydratase activity | IDA PMID:20663849 Functional specification of Arabidopsis isopropylmalate isom... | KEEP AS NON CORE | Summary: He et al. 2010 showed that LeuC interacts with each LeuD (including LeuD2 = SSU3) to form functional IPMIs. Reason: Direct functional evidence for heterodimers containing SSU3 (AtLeuD2). Full text not in cache; the abstract states the LeuC-LeuD heterodimers are functional IPMIs. Subunit-level contribution to the complex activity. This retained heterodimer activity is non-core relative to SSU3 specialization in methionine chain elongation. Supporting Evidence: PMID:20663849 We demonstrate here that AtLeuC physically interacts with AtLeuD proteins to form functional IPMIs. The IPMIs are localized to chloroplast stroma. |
| GO:0003861 3-isopropylmalate dehydratase activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Electronic EC 4.2.1.33 assignment via ARBA/RHEA. Reason: Consistent with the IDA and IBA annotations; the small subunit contributes to the heterodimeric IPMI activity. This retained heterodimer activity is non-core relative to SSU3 specialization in methionine chain elongation. Supporting Evidence: PMID:24608865 On the contrary, IPMI containing SSU2 or SSU3 subunits has a broad substrate range including Met derivatives with different side-chain lengths and IPM. |
| GO:0009098 L-leucine biosynthetic process | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: PAINT inference that the LeuD family acts in L-leucine biosynthesis. Reason: The ancestral LeuD function is Leu biosynthesis, but SSU3 is a Brassicaceae-specific paralog that has been recruited to Met chain elongation. Target-specific evidence shows divergence: the ssu2 ssu3 double knockout does not accumulate IPM and has normal Leu synthesis, whereas SSU1 (LeuD3) is the essential Leu-pathway small subunit. SSU3 can contribute only residually to Leu synthesis when SSU1 is depleted. Not strictly wrong, but it misrepresents the in vivo role of this paralog. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN002879868 · PTN002879868 SUPPORTS SOURCE BUT NOT TARGET Ancestral LeuD node correctly carries Leu biosynthesis (true for SSU1/LeuD3 and microbial LeuD), but the Brassicaceae SSU2/SSU3 duplicates were recruited to Met chain elongation and are dispensable for Leu synthesis. Supporting Evidence: PMID:24608865 Furthermore, IPMI SSU2 and SSU3 play no substantial role in Leu biosynthesis, since IPM does not accumulate to higher levels when both genes are inactivated. PMID:24608865 As indicated by the normal green color a residual Leu biosynthesis occurs in or along the vasculature by the participation of IPMI SSU2 and/or IPMI SSU3. PMID:20663849 The lethal phenotype of the atleud3 mutant suggests functional specification of AtLeuD3 in leucine biosynthesis. PMID:32612621 IPMI SSU2 and IPMI SSU3 participate in the Met chain elongation pathway. |
| GO:0009098 L-leucine biosynthetic process | IEA GO_REF:0000041 | MARK AS OVER ANNOTATED | Summary: UniPathway mapping of L-leucine biosynthesis to SSU3 from its LeuD family membership. Reason: As for the IBA: SSU3 plays no substantial role in Leu biosynthesis in wild-type plants; its physiological role is in Met chain elongation for aliphatic glucosinolates. Supporting Evidence: PMID:24608865 Furthermore, IPMI SSU2 and SSU3 play no substantial role in Leu biosynthesis, since IPM does not accumulate to higher levels when both genes are inactivated. |
| GO:0009507 chloroplast | HDA PMID:25293756 A proteomic strategy for global analysis of plant protein co... | MODIFY | Summary: High-throughput proteomics attributed SSU3 to the chloroplast. Reason: Refine the correct general organelle assignment to chloroplast stroma, an already annotated descendant supported by stromal localization/proteomics. The original source fields are preserved. Proposed replacements: chloroplast stroma Supporting Evidence: PMID:32612621 IPMI SSU1 is found in small plastids, whereas IMPI SSU2 and SSU3 are found in chloroplasts. |
| GO:0009507 chloroplast | ISM GO_REF:0000122 | MODIFY | Summary: AtSubP predicted chloroplast targeting. Reason: Refine the correct general organelle assignment to chloroplast stroma, an already annotated descendant supported by stromal localization/proteomics. The original source fields are preserved. Proposed replacements: chloroplast stroma Supporting Evidence: PMID:19597944 all four IPMI subunits of Arabidopsis are predicted to have chloroplast targeting sequences PMID:32612621 IPMI SSU1 is found in small plastids, whereas IMPI SSU2 and SSU3 are found in chloroplasts. |
| GO:0009536 plastid | IDA PMID:19542295 The plastidic bile acid transporter 5 is required for the bi... | MODIFY | Summary: GFP fused to the N-terminal (transit peptide) region of IPMI1 (= At3g58990, SSU3) localized to plastids in cultured Arabidopsis cells. Reason: Refine the correct general organelle assignment to chloroplast stroma, an already annotated descendant supported by stromal localization/proteomics. The original source fields are preserved. Proposed replacements: chloroplast stroma Supporting Evidence: PMID:19542295 GFP fused to N-terminal fragments of BAT5, IPMI1, IPMI2, and IPMDH1, as well as the plastidic triose phosphate/phosphate translocator, which was used as a positive control, all localized to plastids of cultured Arabidopsis cells PMID:19542295 IPMI1 ( At3g58990 ), IPMI2 ( At2g43100 ) |
| GO:0009536 plastid | IDA PMID:32612621 In Arabidopsis thaliana Substrate Recognition and Tissue- as... | MODIFY | Summary: Full-length SSU3:GFP expressed from its own promoter localizes to plastids (chloroplast-sized) in vascular-associated cells. Reason: Refine the correct general organelle assignment to chloroplast stroma, an already annotated descendant supported by stromal localization/proteomics. The original source fields are preserved. Proposed replacements: chloroplast stroma Supporting Evidence: PMID:32612621 Cross sections through the hypocotyl confirmed that the IPMI SSU3:GFP protein localized to plastids in parenchyma cells surrounding the central vasculature PMID:32612621 IPMI SSU1 is found in small plastids, whereas IMPI SSU2 and SSU3 are found in chloroplasts. |
| GO:0009536 plastid | IEA GO_REF:0000044 | MODIFY | Summary: UniProt subcellular location (Plastid) mapping. Reason: Refine the correct general organelle assignment to chloroplast stroma, an already annotated descendant supported by stromal localization/proteomics. The original source fields are preserved. Proposed replacements: chloroplast stroma Supporting Evidence: PMID:32612621 IPMI SSU1 is found in small plastids, whereas IMPI SSU2 and SSU3 are found in chloroplasts. |
| GO:0009570 chloroplast stroma | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference that plant LeuD subunits are active in the chloroplast stroma. Reason: Supported by direct evidence for stromal localization of LeuC/LeuD IPMIs (He et al. 2010). Supporting Evidence: PMID:20663849 We demonstrate here that AtLeuC physically interacts with AtLeuD proteins to form functional IPMIs. The IPMIs are localized to chloroplast stroma. |
| GO:0009570 chloroplast stroma | IDA PMID:20663849 Functional specification of Arabidopsis isopropylmalate isom... | ACCEPT | Summary: IPMI heterodimers, including those containing LeuD2/SSU3, localized to chloroplast stroma. Reason: Direct experimental evidence (abstract only in cache). Supporting Evidence: PMID:20663849 We demonstrate here that AtLeuC physically interacts with AtLeuD proteins to form functional IPMIs. The IPMIs are localized to chloroplast stroma. |
| GO:0009570 chloroplast stroma | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic stroma assignment via ARBA / UniProt subcellular location. Reason: Consistent with experimental evidence. Supporting Evidence: PMID:20663849 We demonstrate here that AtLeuC physically interacts with AtLeuD proteins to form functional IPMIs. The IPMIs are localized to chloroplast stroma. |
| GO:0019761 glucosinolate biosynthetic process | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA-based electronic assignment of glucosinolate biosynthetic process. Reason: Correct and experimentally supported (He et al. 2010; Imhof et al. 2014; Lachler et al. 2020). Supporting Evidence: PMID:24608865 our metabolite profiling of an ipmi ssu2-1/ipmi ssu3-1 double knockout mutant showed the absolute requirement of these two genes for the biosynthesis of C7 and C8 glucosinolates |
| GO:0019761 glucosinolate biosynthetic process | IMP PMID:20663849 Functional specification of Arabidopsis isopropylmalate isom... | ACCEPT | Summary: atleud1 atleud2 (ssu2 ssu3) mutants show altered aliphatic glucosinolate profiles; LeuD1 and LeuD2 act redundantly in aliphatic glucosinolate biosynthesis. Reason: Core process. SSU3, as part of the IPMI heterodimer with IIL1, catalyzes the isomerization step of the Met chain-elongation cycles; the ssu2 ssu3 double knockout lacks C7/C8 glucosinolates and SSU3 complementation restores them (Imhof et al. 2014). Supporting Evidence: PMID:20663849 Reverse genetics and metabolite profiling showed that AtLeuD1 and AtLeuD2 function redundantly in aliphatic glucosinolate biosynthesis, but AtLeuD3 is not likely to be involved in this pathway. PMID:24608865 our metabolite profiling of an ipmi ssu2-1/ipmi ssu3-1 double knockout mutant showed the absolute requirement of these two genes for the biosynthesis of C7 and C8 glucosinolates PMID:24608865 IPMI SSU3 can catalyze reactions in the formation of C7 and C8 glucosinolates |
| GO:0120528 2-(omega-methylthio)alkylmalate dehydratase activity | EXP PMID:19597944 Arabidopsis thaliana encodes a bacterial-type heterodimeric ... | ACCEPT | Summary: Knill et al. 2009 assigned SSU2 and SSU3 to Met chain elongation (2-(omega-methylthio)alkylmalate isomerization) based on mutant glucosinolate profiles and co-expression with MYB28/MAM1/BCAT4. Reason: Core function. The ssu3-1 single mutant (Ws background) had only a very weak phenotype due to redundancy with SSU2, so the evidence in Knill et al. alone is modest; it is robustly confirmed by the ssu2 ssu3 double knockout, SSU3 complementation, and substrate-recognition-region swaps (Imhof et al. 2014; Lachler et al. 2020). The activity is a property of the LSU1:SSU3 heterodimer, with SSU3 determining substrate specificity. Supporting Evidence: PMID:19597944 Nevertheless the alterations in the glucosinolate composition observed in these mutants suggest that IPMI SSU2 and IPMI SSU3 function in Met chain elongation PMID:19597944 Instead these latter two proteins seem to be functionally redundant as indicated by the relatively weak phenotype of the respective mutants. PMID:19597944 The very weak phenotype of the ipmi ssu3-1 might be due to this mutant being in the Ws-0 background, which per se contains predominantly short-chain glucosinolates PMID:24608865 IPMI SSU3 can catalyze reactions in the formation of C7 and C8 glucosinolates PMID:32612621 Our study shows that the substrate recognition region that differs between IPMI SSU1 and the other two IMPI SSUs determines the substrate preference of IPMI. |
| GO:0120528 2-(omega-methylthio)alkylmalate dehydratase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic EC 4.2.1.170 / RHEA assignment. Reason: Consistent with experimental data on SSU3 in Met chain elongation. Supporting Evidence: PMID:24608865 IPMI SSU3 can catalyze reactions in the formation of C7 and C8 glucosinolates |
| GO:0009316 3-isopropylmalate dehydratase complex | IPI PMID:20663849 Functional specification of Arabidopsis isopropylmalate isom... | NEW | Summary: SSU3 (AtLeuD2) forms a functional heterodimeric IPMI with the large subunit IIL1 (LeuC). Reason: SSU3 is a structural subunit (domain 4, substrate recognition) of the LeuC:LeuD heterodimer defined by GO:0009316; He et al. 2010 demonstrated the physical interaction. Capturing the complex membership conveys that SSU3 contributes to, rather than independently possesses, the isomerase activity. Caveat: Chen et al. 2021 did not detect LeuC-LeuD interaction by Y2H. Supporting Evidence: PMID:20663849 We demonstrate here that AtLeuC physically interacts with AtLeuD proteins to form functional IPMIs. The IPMIs are localized to chloroplast stroma. PMID:32612621 The function of IPMI is defined by the small subunits. |
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Download this section (compressed HTML)Q: What are the kinetic preferences of reconstituted LSU1:SSU3 versus LSU1:SSU2 heterodimers across the C3-C8 2-(omega-methylthio)alkylmalate series, and do the different SRR sequences (YGTLI vs AACTF) confer distinct chain-length preferences?
Q: Is SSU3 a true functional duplicate of SSU2, or do subtle expression (hypocotyl, epidermal) or kinetic differences give it a distinct role?
Q: Should the SSU2/SSU3 contribution to Leu synthesis under SSU1 deficiency be captured in GO, or is L-leucine biosynthesis properly restricted to SSU1 for these paralogs?
Experiment: Purify recombinant LSU1:SSU3 heterodimer and determine kcat/Km with 2-IPM and each 2-(omega-methylthio)alkylmalate (C1-C6 side-chain homologues).
Type: in vitro enzyme kinetics
Experiment: Generate ssu3 single mutants in Col-0 (CRISPR) to remove the Ws background confound and profile aliphatic glucosinolates and chain-elongation intermediates alone and in combination with ssu2.
Type: reverse genetics/metabolomics
Experiment: Co-IP of tagged SSU3 from chloroplasts to test in vivo heterodimerization with IIL1 and possible association with IPMDH1/MAM1.
Type: protein interaction
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