SAGA1 and MITH1 produce matrix-traversing membranes in the CO(2)-fixing pyrenoid.

mith1 mutants lack matrix-traversing membranes, and complementation with MITH1-Venus restores them

"In the complemented strain mith1;MITH1-Venus, an apparently normal matrix-traversing membrane tubule network is restored"
MITH1 is needed to extend the membranes through the matrix, not to initiate them

"These observations suggest that pyrenoid membrane formation initiates at the correct cellular location in the mith1 mutant but then stalls before these membranes are extended through the pyrenoid matrix."
In mith1, thylakoids cross the starch sheath and stop at the matrix surface

"several TEM images of the mith1 mutant showed thylakoid membranes crossing the pyrenoid starch sheath and stopping at the surface of the matrix"
MITH1 localizes to the matrix-traversing membranes, not the matrix as proposed earlier

"Whereas MITH1 was previously proposed to localize to the Chlamydomonas pyrenoid matrix19,20, here we observed that it localizes to and functions at the matrix-traversing membranes."
MITH1 co-pellets with membranes and is released by detergent

"Chlamydomonas cell fractionation also supported membrane localization, as MITH1 co-pelleted with membranes in the absence of detergent and was solubilized when detergent was added"
MITH1 binds SAGA1 in vivo (reciprocal IP-MS)

"SAGA1 was MITH1’s most-abundant specific interactor, and MITH1 was one of SAGA1’s most-specific interactors, indicating that the two proteins physically interact"
No direct MITH1 binding to Rubisco or EPYC1 was detected by yeast two-hybrid

"We could not detect evidence of direct MITH1 physical interaction with Rubisco or EPYC1 by yeast two-hybrid assay"
MITH1 needs SAGA1 for its recruitment to the condensate and for its function; saga1 is epistatic to mith1

"MITH1 requires SAGA1 for its recruitment to the condensate and for its function"
SAGA1 and MITH1 together, but neither alone, make thylakoid membranes traverse a Rubisco condensate in Arabidopsis

"These results demonstrate that SAGA1 and MITH1 together are sufficient to generate matrix-traversing thylakoid membranes in a heterologous system."
Proposed mechanism, MITH1 raises the membrane-matrix adhesive force by binding SAGA1

"We propose that MITH1 increases the membrane–matrix adhesive force per unit area by binding to SAGA1"

Biogenesis, engineering and function of membranes in the CO (2) -fixing pyrenoid.

Preprint summary that MITH1 localizes to the tubules and is needed for their extension

"MITH1 localizes to the tubules and is necessary for their extension through the pyrenoid."

Super-resolution expansion microscopy reveals nanoscale protein domains and CO(2)-dependent remodeling of Chlamydomonas pyrenoid-traversing membranes.

MITH1 is mostly restricted to the cylindrical tubules at the pyrenoid periphery

"MITH1 signal was largely restricted to the cylindrical tubules at the pyrenoid periphery"
The MITH1 C terminus lies away from the tubule membrane, on the matrix side, consistent with an extended coiled coil

"our results suggest that the antibody epitope-containing C-terminus of MITH1 is localized distally from the tubule membrane"
SAGA1 is more peripheral than MITH1, consistent with SAGA1 initiating and MITH1 extending the tubules

"SAGA1 likely acts first at the surface of the pyrenoid to initiate pyrenoid-traversing membranes’ entrance into the matrix, and then MITH1 acts to extend them through the pyrenoid"
MITH1 relocalizes with CO2 level, spread through the network at high CO2 and peripheral at low CO2

"MITH1 was uniformly distributed throughout the tubule network at high CO2 and became increasingly peripheral during the transition to low CO2."
The authors cite unpublished/preprint work reporting that MITH1 binds membranes and Rubisco

"We recently showed that MITH1 can bind both membranes and Rubisco, likely serving to link the pyrenoid tubule and matrix compartments40."

PMID:35513725

Systematic characterization of gene function in the photosynthetic alga Chlamydomonas reinhardtii.

Cre06.g259100 was named SAGA3 for its homology to SAGA1 and SAGA2, from a genome-wide mutant phenotyping screen

"We named one of these genes, Cre06.g259100, SAGA3 (STARCH GRANULES ABNORMAL FAMILY MEMBER 3) because its protein product shows homology to the two pyrenoid structural proteins SAGA1 and SAGA2"
SAGA3 localizes to the pyrenoid

"Consistent with a role in the CCM, SAGA3 localizes to the pyrenoid"

PMID:28938113

A Spatial Interactome Reveals the Protein Organization of the Algal CO(2)-Concentrating Mechanism.

Cre06.g259100 was earlier listed as a pyrenoid matrix protein of unknown function

"a protein of unknown function (Cre06.g259100; Kobayashi et al., 2016)"

PMID:33177094

Assembly of the algal CO(2)-fixing organelle, the pyrenoid, is guided by a Rubisco-binding motif.