HLA3 (Chlamydomonas reinhardtii) — curation notes

UniProt A0A2K3E226 (TrEMBL, unreviewed; ProtNLM name "ABC transporter, multidrug
resistance associated protein"); locus Cre02.g097800 (CHLRE_02g097800v5). Synonym
CrMRP1. 1325 aa.

Deep research

• 2026-10-03: scripts/deep_research_wrapper.py CHLRE HLA3 falcon --fallback perplexity-lite
  failed. Falcon returned HTTP 402 Payment Required; the perplexity-lite fallback failed
  because the perplexity provider was not available in this environment. Not retried
  (per instructions). Notes below are from primary literature read directly from the
  publications/ cache.

Sequence / domain features (from HLA3-uniprot.txt, plus a quick motif check)

• Full-size ABCC/MRP architecture: TMD1 (166-384), NBD1 (418-639), TMD2 (711-1003),
  NBD2 (1062-1297); PANTHER PTHR24223:SF443 (ABCC subfamily), CDD ABC_6TM_ABCC_D1/D2 and
  ABCC_MRP_domain1/2; ~12+ predicted TM helices (Phobius). There is no N-terminal TMD0
  in InterPro/CDD, so it resembles the "short" MRPs (MRP4/5-like) rather than MRP1.
• Motif inspection of the UniProt sequence (done inline, not a deposited analysis):
  NBD1 Walker A GRIAAGKS, NBD1 Walker B LVLLDN (catalytic Glu replaced by Asn),
  NBD1 signature FSGGQ (canonical); NBD2 Walker A GRTGSGKST, NBD2 Walker B ILCLDE
  (canonical), NBD2 signature SLGQ (degenerate). This is the asymmetric
  one-consensus / one-degenerate ATP-site arrangement typical of ABCC proteins; the
  consensus site (NBD2 Walker A/B + NBD1 signature) is intact, so ATP binding and
  hydrolysis are expected.

Expression and regulation

• Limiting-CO2 inducible; only one of seven Chlamydomonas MRP-type genes controlled by
  CIA5 PMID:19321421.
• In the CIA5/CO2 RNA-seq study HLA3 falls in CCM cluster 15 with LCIA, LCI1 and CCP1
  PMID:22634760.
• HLA3 mRNA is reduced in LCIA insertion mutants; LCIA is unaffected in the HLA3 mutant,
  suggesting a chloroplast-to-nucleus signal PMID:26015566.

Localization

• Originally only predicted (WoLF PSORT) to be plasma membrane PMID:19321421.
• Direct: anti-HLA3 immunofluorescence at the cell periphery and enrichment in purified
  plasma membrane fractions [PMID:26015566 "a notable enrichment of HLA3 was observed in the
  PM fraction"; "we concluded that HLA3 and LCIA were localized to the PM and CE,
  respectively"].
• Venus-tagged HLA3 at the plasma membrane in Chlamydomonas, and GFP-tagged HLA3 at the
  plasma membrane in tobacco leaf cells PMID:26538195.
• UniProt ARBA (ARBA00004128) and the GO:0005774 vacuolar membrane IEA are therefore
  contradicted by direct data (the rule is a family-level transfer from plant vacuolar
  ABCC/MRP glutathione-conjugate pumps).

Function

• RNAi knockdown (2 constructs): HLA3 knockdown alone gives modest, high-pH-specific loss of
  Ci affinity and Ci uptake in very-low CO2; combined with LCIB mutation or off-target LCIA
  co-knockdown, dramatic loss of growth and Ci uptake at pH 9 PMID:19321421. The authors
  explicitly leave open direct vs. indirect action PMID:19321421.
• dTALE activation of HLA3 in high-CO2 cells increased Ci accumulation and Ci-dependent O2
  evolution at very low CO2 [PMID:25660294 abstract: "confirms that HLA3 is indeed involved
  in Ci uptake, and suggests it is mainly associated with HCO3(-) transport"]. Abstract only
  in cache.
• Insertion mutant Hin-1 (complemented by Hin-1C): reduced Ci affinity at pH 9 and lower
  [14C]-Ci accumulation PMID:26015566. HLA3/LCIA double mutant additive. Single HLA3
  overexpression in HC gave only a small Ci accumulation increase; co-overexpression with LCIA
  significantly raised Ci affinity/accumulation PMID:26015566.
• Heterologous: HLA3 (untagged or GFP-tagged) at the oocyte surface raised H14CO3- uptake
  2.7-fold over water-injected controls PMID:26538195. The
  authors caution the mode (active vs passive) is unresolved PMID:26538195. This is the most direct evidence that HLA3 itself moves
  bicarbonate across a membrane; no ATP-dependence, substrate specificity or purified
  protein data exist.
• Interactome: HLA3 co-purifies reciprocally with LCI1 and with the P-type ATPase ACA4,
  plus a CaM kinase and CYG63 cyclase as high-confidence interactors PMID:28938113. Not a GO
  complex; recorded as a question only. Blue-native PAGE shows HLA3 in a ~580 kDa complex
  independent of LCIA PMID:26015566.

Curation decisions summary

• MF: GO:0015106 bicarbonate transmembrane transporter activity (NEW; oocyte uptake +
  in vivo genetics), keep IBA GO:0140359 ABC-type transporter activity; ATP binding and ATP
  hydrolysis IEA accepted (consensus ATP site intact).
• CC: GO:0005886 plasma membrane (NEW; IDA). REMOVE GO:0005774 vacuolar membrane (IEA, ARBA)
  — contradicted.
• BP: GO:0015701 bicarbonate transport (NEW); keep transmembrane transport IBA/IEA.
• Did not add a CCM-level or "response to CO2" process: induction by low CO2 is regulation of
  the gene, not activity of the product.