HLA3 (High Light Activated 3, also CrMRP1; locus Cre02.g097800) is a full-size ATP-binding cassette transporter of the ABCC (multidrug resistance-associated protein, MRP) subfamily in the green alga Chlamydomonas reinhardtii. It has two transmembrane domains and two nucleotide-binding domains, with one consensus and one degenerate ATP site as is typical of ABCC proteins. HLA3 is one of seven MRP-type genes in the genome but the only one induced by limiting CO2 under control of the CCM master regulator CIA5, and its protein accumulates within hours of transfer to very low CO2. Immunofluorescence, plasma membrane fractionation and fluorescent-protein tagging place HLA3 in the plasma membrane. Loss of HLA3 (RNAi knockdown or insertional mutation) reduces photosynthetic inorganic carbon affinity and intracellular inorganic carbon accumulation, mainly at alkaline pH where bicarbonate is the dominant species, and the defect is strongly enhanced by loss of the chloroplast envelope protein LCIA or the stromal protein LCIB. Ectopic expression raises cellular inorganic carbon accumulation, most strongly when co-expressed with LCIA, and HLA3 expressed in Xenopus oocytes increases bicarbonate uptake. HLA3 therefore supplies external bicarbonate across the plasma membrane as the first step of the algal CO2-concentrating mechanism, acting with LCIA, which moves bicarbonate on into the chloroplast stroma. Whether HLA3 transports bicarbonate itself by an ATP-driven mechanism has not been shown biochemically. In the spatial interactome HLA3 associates with the plasma membrane protein LCI1 and the P-type ATPase ACA4.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: HLA3 has two ABC nucleotide-binding domains; NBD2 carries canonical Walker A and Walker B motifs and NBD1 a canonical ABC signature, so nucleotide (ATP) binding is expected. Reason: Correct but uninformative parent of ATP binding (GO:0005524), which is also annotated. Nucleotide binding is a sub-step of the ABC transport cycle, not the core function. |
| GO:0005524 ATP binding | IEA GO_REF:0000120 | ACCEPT | Summary: Full-size ABCC transporter with two NBDs (residues 418-639 and 1062-1297). Inspection of the sequence shows NBD1 Walker A GRIAAGKS and NBD2 Walker A GRTGSGKST, with an intact consensus ATP site formed by the NBD2 Walker A/B and the NBD1 LSGGQ-type signature (FSGGQ). Reason: Domain-based inference is sound; the ATP-binding motifs are conserved. ATP binding is intrinsic to the ABC-type transporter activity accepted below. No direct nucleotide-binding data exist for HLA3. |
| GO:0005774 vacuolar membrane | IEA GO_REF:0000044 | REMOVE | Summary: Derived from the UniProt ARBA rule ARBA00004128 (Vacuole membrane), a family-level transfer typical of plant vacuolar ABCC/MRP conjugate pumps. Direct experiments place HLA3 in the plasma membrane. Reason: Contradicted by direct evidence in Chlamydomonas. Anti-HLA3 immunofluorescence is peripheral and HLA3 is enriched in purified plasma membrane fractions (PMID:26015566), and Venus-tagged HLA3 is in the plasma membrane in Chlamydomonas and tobacco (PMID:26538195). No study reports a vacuolar pool. The correct location (GO:0005886 plasma membrane) is proposed as a NEW annotation below. Supporting Evidence: PMID:26015566 From these results, we concluded that HLA3 and LCIA were localized to the PM and CE, respectively. PMID:26538195 LCI1: Venus and HLA3: Venus were in the plasma membrane. |
| GO:0016020 membrane | IEA GO_REF:0000002 | ACCEPT | Summary: HLA3 is a polytopic integral membrane protein (two ABC transmembrane domains, multiple predicted TM helices). Reason: Correct, if general. The specific location is the plasma membrane, added as a NEW annotation. |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000002 | ACCEPT | Summary: HLA3 retains one consensus ATP site (NBD2 Walker A and Walker B with the catalytic glutamate, plus the NBD1 signature), while NBD1 Walker B has the asparagine substitution typical of the degenerate site of ABCC proteins. ATP hydrolysis by the consensus site is expected. Reason: Supported by conserved catalytic motifs in a full-size ABC transporter. ATPase activity has not been measured for HLA3, and whether bicarbonate uptake via HLA3 is ATP-driven is untested (PMID:26538195 leaves active versus passive transport open), so this stays a sequence-based inference. Supporting Evidence: PMID:26538195 Further kinetic analyses will be required to determine the mode of action of these proteins (i.e. active vs. passive transport). |
| GO:0055085 transmembrane transport | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference at the ABCC family node (PTN000657997). HLA3 is a full-size ABCC member and genetics, gain-of-function and oocyte data show it supports bicarbonate movement across the plasma membrane. Reason: Correct at the family level and consistent with HLA3 biology. The more specific process, bicarbonate transport (GO:0015701), is proposed as a NEW annotation. Supporting Evidence: PMID:26015566 These results indicated that HLA3 has a meaningful role in HCO3– uptake in VLC conditions. |
| GO:0055085 transmembrane transport | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the ABC transmembrane type-1 domain. Reason: Correct and consistent with the IBA and with experimental evidence for a role in bicarbonate uptake. |
| GO:0140359 ABC-type transporter activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference for the ABCC clade (PTN009085477). HLA3 has the complete TMD-NBD-TMD-NBD architecture of the clade and an intact consensus ATP site, with no evidence of loss of transport function. Reason: HLA3 lies within the ABCC clade and retains the features that define ABC-type transport; nothing argues for loss or divergence of the ATPase-coupled mechanism. The term is generic about substrate; the bicarbonate specificity suggested by experiments is captured by the NEW GO:0015106 annotation. Direct proof that HLA3-dependent bicarbonate uptake is ATP-coupled is still lacking. |
| GO:0140359 ABC-type transporter activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the ABC transmembrane and ABCC 6TM domains. Reason: Consistent with the IBA annotation and the domain architecture. |
| GO:0005886 plasma membrane | IDA PMID:26015566 Characterization of cooperative bicarbonate uptake into chlo... | NEW | Summary: Anti-HLA3 immunofluorescence gives a peripheral signal, and HLA3 is enriched with the plasma membrane markers LCI1 and H+-ATPase in purified plasma membrane fractions. Venus-tagged HLA3 is also at the plasma membrane in Chlamydomonas, and GFP-tagged HLA3 at the plasma membrane of tobacco cells and Xenopus oocytes. Reason: Direct, consistent localization by two independent groups and three methods. There is no GOA plasma membrane annotation; the only GOA location (vacuolar membrane) is an ARBA transfer contradicted by these data. This is the membrane across which HLA3 acts. Supporting Evidence: PMID:26015566 Similarly, a notable enrichment of HLA3 was observed in the PM fraction. LCIA was highly enriched in the CE fraction, where CE protein CCP1 (30) was also enriched. From these results, we concluded that HLA3 and LCIA were localized to the PM and CE, respectively. PMID:26538195 LCI1: Venus and HLA3: Venus were in the plasma membrane. |
| GO:0015106 bicarbonate transmembrane transporter activity | IDA PMID:26538195 Introducing an algal carbon-concentrating mechanism into hig... | NEW | Summary: HLA3 expressed in Xenopus oocytes reaches the cell surface and increases H14CO3- uptake 2.7-fold over controls. In Chlamydomonas, loss of HLA3 reduces Ci accumulation mainly at alkaline pH where bicarbonate dominates, and ectopic HLA3 expression raises Ci accumulation, most strongly with LCIA. Reason: The heterologous oocyte assay shows HLA3 itself confers bicarbonate uptake across a membrane, and it agrees with the pH-dependence of the knockdown and insertion mutant phenotypes (PMID:19321421, PMID:26015566) and with gain-of-function data (PMID:25660294). The participation test is met because HLA3 is the membrane protein proposed to move the substrate, not a regulator of another transporter. Caveats are that the oocyte effect is modest, bicarbonate versus CO2 specificity and ATP-dependence were not tested, and Duanmu et al. could not exclude an indirect role. GO has no ABC-type bicarbonate term, so this activity is recorded alongside GO:0140359. Supporting Evidence: PMID:26538195 Oocytes transformed with mLCIA: GFP or HLA3: GFP accumulated 2.0‐ and 2.7‐fold more 14C than water‐injected controls, respectively PMID:26015566 These results indicated that HLA3 has a meaningful role in HCO3– uptake in VLC conditions. PMID:25660294 Activation of HLA3 expression in high CO2 acclimated cells, where HLA3 is not expressed, resulted in increased Ci accumulation and Ci-dependent photosynthetic O2 evolution specifically in very low CO2 concentrations |
| GO:0015701 bicarbonate transport | IMP PMID:26015566 Characterization of cooperative bicarbonate uptake into chlo... | NEW | Summary: HLA3 insertion mutant (Hin-1) has reduced Ci affinity at pH 9 and lower [14C]-Ci accumulation, restored by complementation; RNAi knockdown gives high-pH-specific loss of Ci uptake that is greatly enhanced when LCIA or LCIB is also lost. Reason: The process is the direct consequence of the transporter activity above, at the plasma membrane; HLA3 does the work of moving the substrate rather than merely being required. It refines the generic transmembrane transport IBA/IEA. A broader CO2-concentrating mechanism or response-to-CO2 process is not proposed, since induction by low CO2 describes regulation of the gene, not the activity of the product. Supporting Evidence: PMID:26015566 These results indicated that HLA3 has a meaningful role in HCO3– uptake in VLC conditions. PMID:19321421 Collectively, the data presented here provide compelling evidence that HLA3 is directly or indirectly involved in HCO 3 − transport |
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Download this section (compressed HTML)Q: Is HLA3 itself a primary-active, ATP-driven bicarbonate pump, or does it facilitate bicarbonate entry indirectly (for example as a channel-like ABCC or a regulator of LCI1/ACA4), given that only a 2.7-fold oocyte uptake effect and no ATPase or substrate-specificity data exist?
Suggested experts: Spalding MH, Fukuzawa H
Q: Should GO provide an ABC-type bicarbonate transporter activity term (as a child of both GO:0140359 and GO:0015106) for HLA3 and cyanobacterial CmpABCD-type transporters?
Q: What is the composition and function of the plasma membrane complex containing HLA3, LCI1 and the P-type ATPase ACA4 seen by AP-MS, and does ACA4 energise HLA3-dependent uptake?
Suggested experts: Mackinder LCM
Experiment: Express wild-type HLA3 and Walker B (E to Q in NBD2) or Walker A (K to M) mutants in Xenopus oocytes or yeast and measure H14CO3- uptake, ATP dependence (vanadate, ATP depletion) and CO2 versus bicarbonate preference; complement the Hin-1 mutant with the same variants.
Hypothesis: HLA3 transports bicarbonate by an ATP-hydrolysis-coupled mechanism through its consensus ATP site.
Type: heterologous transport assay and genetic complementation
Experiment: Purify HLA3 in detergent or nanodiscs, measure basal and bicarbonate-stimulated ATPase activity, and reconstitute into proteoliposomes for vectorial bicarbonate transport.
Hypothesis: Purified HLA3 has bicarbonate-stimulated ATPase activity.
Type: biochemistry
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