Protein import into complex plastids
Final Report Abstract
In the funding period we dealt with six aspects of the protein import into complex plastids including its evolutionary implications: (i) Based on preliminary work, we finally published that protein transport by the help of a SELMA complex is depending on lysines in the N-terminal regions of nucleus-encoded proteins imported into the PPC and stroma. In addition, we showed that the elimination of the N-terminal lysines effects viability of the cell. (ii) We have performed in silico/in vivo localization screens for PPM protein candidates. Two aspects were of special interest: First, an already identified PPM located rhomboid protease was used for protein-protein interaction studies indicating that an E3 ligase is part of the SELMA complex. Second, identified mitochondrial carrier proteins showed a targeting from the PPC into plastid surrounding membranes; a phenomenon so far not know form diatoms. In addition, in pull down/mass spectrometry/in vivo localization screens we additionally identified further candidate proteins, which might be involved in the targeting of PPC and stroma proteins. (iii) Our present results showed that homologs to the UPR factors IRE1 and PERK are encoded by the diatom. According to a model, these UPR factors should be restricted to the host ER but not to the cER, both having a continuous lumen. This hypothesis is tested at the moment in colocalization studies by the use of eGFP and mRuby3 fusion proteins. (iv) Using a protocol established in my lab, we applied the CRISPR /Cas9 method to knockout SELMA factors. However, as these factors are most likely essential for viability, a strategy was developed to complement putative knockouts by a rescue protein, which is equal in the amino acid sequence to the wild type protein but the gene is not a target for CRISPR/Cas9 actions. (v) Together with the groups of S. Gould and Bill Martin I theoretically analyzed the fact that a SELMA complex is present in organisms with a complex red algal derived plastid (with the exception of peridinin-containing dinoflagellates) investigated so far. We concluded that the common presence of SELMA indicates a single origin of these organisms and that the both outermost plastid membranes of complex plastids are of ER origin. (vi) Finally, we investigated if the SELMA complex is, as proposed by T. Cavalier-Smith, located in a PPC specific internal compartment. However, ultrastructural analyses in addition to screens for vesical generating and fusion proteins as well as for compartment-specifying proteins did not support the Cavalier-Smith model.
Publications
- (2015) N-terminal lysines are essential for protein translocation via a modified ERAD system in complex plastids. Mol Microbiol 96:609-620
Lau JB, Moog D, Stork S, Sommer MS, Maier UG
(See online at https://doi.org/10.1111/mmi.12959) - (2015) Protein import and the origin of red complex plastids. Current Biology 25:R515–R521
Gould SB, Maier UG, Martin WF
(See online at https://doi.org/10.1016/j.cub.2015.04.033) - (2016) Protein-protein interactions indicate composition of a 480 kDa SELMA complex in the second outermost membrane of diatom complex plastids. Mol Microbiol 100:76-89
Lau JB, Stork S, Moog D, Schulz J, Maier UG
(See online at https://doi.org/10.1111/mmi.13302) - (2017) Cellular compartmentation follows rules: The Schnepf theorem, its consequences and exceptions: A biological membrane separates a plasmatic from a non-plasmatic phase. BioEssays, 39(8)
Moog D, Maier UG
(See online at https://doi.org/10.1002/bies.201700030) - (2017) Origin of complex algae by secondary endosymbiosis: A journey through time. Protoplasma 254: 1835-1843
Gentil J, Hempel F, Moog D, Zauner S, Maier UG
(See online at https://doi.org/10.1007/s00709-017-1098-8) - (2019) The known, the new, and a possible surprise. A re-evaluation of the nucleomorph-encoded proteome of cryptophytes. Genome Biol Evol 11:1618-1629
Zauner S, Heimerl T, Moog D, Maier UG
(See online at https://doi.org/10.1093/gbe/evz109)
