{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,4]],"date-time":"2025-11-04T05:37:49Z","timestamp":1762234669371,"version":"build-2065373602"},"reference-count":83,"publisher":"MDPI AG","issue":"21","license":[{"start":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T00:00:00Z","timestamp":1761868800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NIH","award":["R15GM137251"],"award-info":[{"award-number":["R15GM137251"]}]},{"name":"the Ernest H. Volwiler Professorship (CDS) at Miami University"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Plants"],"abstract":"<jats:p>Tha4, the smallest component of the cpTAT system, is thought to be the pore-forming element in the TAT translocase. A conserved glutamate at the 10th position in its transmembrane helix is crucial for function. Substitution of this glutamate with alanine abolishes transport, while aspartate substitution partially restores it, highlighting the importance of charge and hydrophobicity. To examine these effects, we generated Tha4 variants with different glutamate substitutions and assessed their transport abilities. Additionally, we developed assays to evaluate Tha4 oligomerization in the presence or absence of a proton motive force (PMF) and functional precursor proteins. Glutamate positional substitutions designed to increase proximity to the acidified lumen were not tolerated in the alanine background, whereas aspartate variants showed slight tolerance. Oligomerization assays revealed that Tha4 oligomer formation in the transmembrane helix region was primarily dependent on the presence of a functional precursor, regardless of PMF, while C-tail oligomer formation responded mainly to PMF. The amphipathic region showed no significant response to either factor. Alanine substitution enhanced oligomerization, while aspartate reduced it, likely due to altered packing interactions between monomers. These discoveries highlight the crucial function of the transmembrane glutamate in sustaining Tha4 activity and ensuring appropriate assembly during activation transport.<\/jats:p>","DOI":"10.3390\/plants14213338","type":"journal-article","created":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T14:00:33Z","timestamp":1762178433000},"page":"3338","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["The Transmembrane Glutamate Serves as a pH Sensor for Tha4 Oligomerization During Twin Arginine Transport of Proteins"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-4823-1202","authenticated-orcid":false,"given":"Vidusha S.","family":"Weesinghe","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6928-8802","authenticated-orcid":false,"given":"Christopher Paul","family":"New","sequence":"additional","affiliation":[{"name":"Cellular, Molecular, and Structural Biology Graduate Program, Miami University, Oxford, OH 45056, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1347-3870","authenticated-orcid":false,"given":"Carole","family":"Dabney-Smith","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA"},{"name":"Cellular, Molecular, and Structural Biology Graduate Program, Miami University, Oxford, OH 45056, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1105\/tpc.12.3.319","article-title":"Proteomics of the chloroplast: Systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins","volume":"12","author":"Peltier","year":"2000","journal-title":"Plant Cell"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1016\/0092-8674(89)90898-2","article-title":"Transport and Routing of Proteins into Chloroplasts","volume":"56","author":"Keegstra","year":"1989","journal-title":"Cell"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"332","DOI":"10.1016\/j.bbamcr.2012.03.019","article-title":"Molecular chaperone involvement in chloroplast protein import","volume":"1833","author":"Jarvis","year":"2013","journal-title":"Biochim. 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