MITH1 (Chlamydomonas reinhardtii) curation notes

UniProt A0A2K3DMK6 (TrEMBL, "Uncharacterized protein"). Locus Cre06.g259100
(CHLRE_06g259100v5). 1,304 aa, 138,446 Da predicted. Aliases are SAGA3
(Fauser et al. 2022) and MITH1, MIssing THylakoids 1 (Hennacy et al. 2024).

GOA had 0 rows at review time, so every annotation in the review is a NEW
proposal.

Deep research

Deep research was not run. The falcon provider returns HTTP 402 and perplexity
is not available in this environment, so the wrapper was skipped as
instructed. The review rests on the cached primary literature below. There is
no -deep-research-*.md file for this gene.

Sequence features (UniProt record)

• Long predicted coiled-coil segments: 208-313, 396-633, 683-710 and 739-990
  (SAM:Coils). There is also a Tropomyosin-like SUPFAM hit (SSF57997).
• Disordered, low-complexity and Gly-rich regions at 60-180, 1194-1222 and
  1258-1304 (MobiDB-lite).
• No CBM20 starch-binding domain, unlike SAGA1 and SAGA2.
• No predicted transmembrane segment.
• No detectable Rubisco-binding motif PMID:39548241.
• The PANTHER PTHR43941 "structural maintenance of chromosomes protein 2" hit
  probably reflects only the generic long coiled-coil. There is no evidence of
  SMC-like function, so it is ignored.

Literature summary

Mackinder et al. 2017 (PMID:28938113)

• Lists Cre06.g259100 as a pyrenoid matrix protein of unknown function
  PMID:28938113. This was superseded by the
  tubule localization below.

Fauser et al. 2022, Nat Genet (PMID:35513725)

• A genome-wide mutant screen found that the gene is needed for normal growth
  at low CO2 and named it SAGA3 for its homology to SAGA1 and SAGA2
  PMID:35513725.
• It localizes to the pyrenoid PMID:35513725.

Hennacy et al. 2024, Nat Plants (PMID:39548241; preprint PMID:39211136)

• Mutant phenotype and complementation. mith1 pyrenoids lack matrix-traversing
  membranes, and MITH1-Venus restores them PMID:39548241.
• Extension, not initiation. Membranes start at the right place but stall at
  the matrix surface PMID:39548241.
• Localization. MITH1 is on the matrix-traversing membranes, not in the matrix
  PMID:39548241. It co-pellets with membranes
  and is released by detergent PMID:39548241.
• Interactions:
• Reciprocal IP-MS shows that MITH1 and SAGA1 bind PMID:39548241.
• No yeast two-hybrid interaction with Rubisco or EPYC1 was found
  PMID:39548241.
• Epistasis and recruitment. saga1 is epistatic to mith1, and MITH1 needs SAGA1
  to localize PMID:39548241.
• Heterologous sufficiency. SAGA1 and MITH1 together, but neither alone, make
  thylakoid sheets traverse an EPYC1-Rubisco condensate in Arabidopsis
  PMID:39548241. The membranes are sheets, not tubules, so other
  factors shape the tubules.
• Model. MITH1 raises the membrane-matrix adhesion that SAGA1 provides
  PMID:39548241.
• Phenotype severity. mith1 grows less poorly than saga1 at air CO2 and has
  fewer extra condensates. CAH3 is found at pyrenoid-peripheral puncta in
  mith1.

Expansion microscopy preprint, 2026 (PMID:42465278)

• MITH1 is mostly on the peripheral, cylindrical tubules PMID:42465278.
• The MITH1 C terminus is distal to the membrane, consistent with an extended
  coiled coil reaching into the matrix PMID:42465278.
• SAGA1 lies more peripheral than MITH1, which fits SAGA1 initiating the
  tubules and MITH1 extending them.
• MITH1 relocalizes with CO2 level PMID:42465278.
• The authors cite their own unpublished or preprint structural work (ref. 40)
  for MITH1 binding membranes and Rubisco PMID:42465278. That work is not in PubMed
  (esearch "MITH1 AND pyrenoid" returns only 39548241, 39211136 and 42465278),
  so it could not be checked. It also conflicts with the negative yeast
  two-hybrid result.

Other cached papers

• Meyer et al. 2020 (PMID:33177094) defines the Rubisco-binding motif. MITH1
  is not discussed, and Hennacy cites it for MITH1 lacking the motif.
• Crans et al. 2026 (PMID:42090253), on SAGA1/SAGA2 and the starch sheath,
  does not discuss MITH1. Not cited in the review.

Decisions

1. Location, GO:0160223 pyrenoid tubule (NEW, IDA). Strongly supported by a
   functional fusion, membrane fractionation and antibody-based expansion
   microscopy. No separate GO:1990732 pyrenoid row, because the tubule term is
   part_of pyrenoid.
2. Process, GO:0010027 thylakoid membrane organization (NEW, IMP).
3. Participation test. MITH1 is physically on the membranes being
   reorganised, binds SAGA1, and is proposed to supply part of the adhesive
   force that moves the membrane into the matrix. That is a structural
   contribution to the step, not substrate-like necessity.
4. The evidence is complemented loss of function plus heterologous
   sufficiency with SAGA1.
5. Comparator check. VIPP1 and FZL (Arabidopsis thylakoid shapers) carry
   GO:0010027, and SAGA1 was given the same term in its sibling review.
6. There is no "pyrenoid tubule assembly" term. Requesting one is raised as a
   suggested question and an ontology gap.
7. Molecular function, GO:0043495 protein-membrane adaptor activity (core
   function only, tentative). The term matches the SAGA1 annoton. MITH1
   associates with membranes and binds SAGA1 (a Rubisco binder), bridging SAGA1
   and the matrix to the membrane surface.
8. Caveats: there is no membrane-binding domain, lipid binding has not been
   measured, and direct Rubisco binding is disputed.
9. It is not added as a NEW existing-annotation row, because no direct assay
   of the activity exists. The validator warns about this, and the warning is
   accepted.
10. "Protein binding" was avoided.
11. Not proposed:
12. Starch binding: MITH1 has no CBM20.
13. Rubisco binding: the yeast two-hybrid result is negative, and the only
    positive report is an uncached, unverifiable citation.
14. Any CCM or CO2-response process term: the low-CO2 growth defect is a
    downstream consequence of missing tubules.

Proposed module annoton (for modules/pyrenoid_ccm.yaml, node tubule_matrix_tethering)

• id: mith1_tubule_extension
• participant: UniProtKB:A0A2K3DMK6 (Chlamydomonas MITH1 / SAGA3, Cre06.g259100)
• function: GO:0043495 protein-membrane adaptor activity (tentative)
• process: GO:0010027 thylakoid membrane organization
• location: GO:0160223 pyrenoid tubule
• role: recruited by SAGA1, and needed to extend the tubules through the matrix
  after SAGA1 has started them.