About

Steven M. Theg is a Distinguished Professor Emeritus in Plant Biology at the College of Biological Sciences, UC Davis. His research focuses on protein translocation across biological membranes, with particular emphasis on chloroplast membranes. Theg's laboratory aims to understand the events surrounding the transport of proteins into chloroplasts and their assembly into multimeric complexes. Most of his work involves studying protein trafficking and assembly in chloroplasts isolated from higher plants such as peas, Arabidopsis, and N. benthamiana, which serve as effective model systems due to their ease of organelle separation and high metabolic activity post-isolation. His research also explores the mechanisms of thylakoid division and stromule formation, contributing to the broader understanding of cellular protein targeting and organelle biochemistry. Theg's work has implications for understanding protein targeting paradigms that are utilized by various organelles, and his findings have advanced knowledge in plant cell biology and membrane protein trafficking.

Research topics

• Biology
• Chemistry
• Biophysics
• Biochemistry
• Cell biology

Selected publications

• Molecular dynamic studies on the interaction of a TatA oligomer with Tat translocon substrates

  Biochimica et Biophysica Acta (BBA) - Biomembranes · 2026-02-24

  articleSenior author
  PublisherDOI

• Author response for "From cytoplasm to lumen—mapping the free pools of protein subunits of three photosynthetic complexes using quantitative mass spectrometry"

  2025-02-14

  peer-review
  PublisherDOI

• Molecular Dynamic Studies on the Interaction of a TatA Oligomer with Tat Translocon Substrates

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-09-30

  preprintOpen accessSenior author

  Abstract The Tat Translocon directly utilizes the Proton Motive Force to transport folded proteins from the n-side to the p-side of energized membranes, targeting the thylakoid lumen of chloroplasts and the periplasmic space of Bacteria and Archaea. In most organisms the Translocon consists of three subunits, TatA, TatB and TatC exhibiting a stoichiometry of ∼20-50/1/1. While TatB/TatC recognize the canonical twin-arginine motif - containing signal sequence of substrate proteins, TatA has been hypothesized to interact with TatB/TatC and translocon substrates facilitating their transport across the membrane. TatA from E.coli contains a short transmembrane helix near the N-terminus, a longer amphipathic helix and a relatively large unstructured C-terminal domain. While the transmembrane and amphipathic helixes are required for Translocon activity, the C-terminal domain is, in large measure, dispensable. TatA has been hypothesized to form higher-order oligomers in the biological membranes. In this communication we have used 1000 ns-long course-grained molecular dynamic simulations to examine the interactions between a membrane-associated E. coli TatA nonamer, alone, and in association with two Tat Translocon substrate proteins, either OEE17 or TorA. In all simulations, either in the presence or absence of substrate, the TatA nonamer markedly thinned the lipid bilayer which may facilitate substrate translocation. The pore of the nonamer was occupied by a phospholipid layer consisting of ∼6 phospholipids in the absence of substrates and ∼11 phospholipids in their presence. Structurally, the amphipathic helix of TatA were observed to exhibit significant conformational flexibility which appears to facilitate TatA-substrate interactions. In the absence of substrate the TatA nonamer was unstable with its radial architecture collapsing in 200-300 ns. In the presence of substrate, however, the radial geometry of the nonamer persists for at least 1000 ns. Interestingly, in the presence of the smaller substrate OEE17, fewer TatA monomers are retained in a radial geometry then observed in the presence of the larger substrate TorA indicating that the molecularity of the TatA oligomer can adjust to the size of the substrate. Specific hydrophilic residues of the TatA amphipathic helixes were found to interact with both substrate molecules, and these form quite stable charge-pair or hydrogen-bonding interactions. While the substrate proteins were initially placed adjacent to the amphipathic helixes of the nonamer, during the simulation trajectories the substrates moved to a more central position adjacent to, and partially entering, the oligomer pore. Concomitantly, the oligomer was observed to lose phospholipids. These latter observations may constitute a glimpse of the initial stages of protein translocation.

  PublisherOA PDFDOI

• From cytoplasm to lumen—mapping the free pools of protein subunits of three photosynthetic complexes using quantitative mass spectrometry

  FEBS Letters · 2025-03-12

  articleOpen access

  The phycobilisome (PBS) captures light energy and transfers it to photosystem I (PSI) and photosystem II (PSII). Which and how many copies of protein subunits in PBSs, PSI, and PSII remain unbound in thylakoids are unknown. Here, quantitative mass spectrometry (QMS) was used to quantify substantial pools of free extrinsic subunits of PSII and PSI. Interestingly, the membrane intrinsic PsaL is 3-fold higher than PsaA/B. This scenario complements the static structures of these complexes as revealed by X-ray crystallography and cryo-EM. The ratios of ApcG and photoprotective OCP over PBS indicate a pool of extra ApcG. The 2.5 ratio of CpcG-PBS over CpcL-PBS improves our understanding of these light-harvesting complexes involved in energy capture and photoprotection in cyanobacteria. Impact statement Our study presents the first quantitative inquiry of the free pools of proteins associated with the three major photosynthetic complexes in Synechocystis 6803. This study increases our understanding of the unbound thylakoid proteome, guiding future research into the functions of these proteins, which will facilitate efforts to enhance photosynthetic efficiency.

  PublisherOA PDFDOI

• A real-time analysis of protein transport via the twin arginine translocation pathway in response to different components of the protonmotive force

  Journal of Biological Chemistry · 2023-09-22 · 6 citations

  articleOpen accessSenior authorCorresponding

  The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the transmembrane electrical potential (Δψ) component of PMF in vitro. To investigate the comprehensive PMF requirement in Escherichia coli, we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology in E. coli spheroplasts. This luminescence assay allows for continuous monitoring of Tat transport with high-resolution, making it possible to observe subtle changes in transport in response to different treatments. By applying the NanoLuc assay, we report that, under acidic conditions (pH = 6.3), ΔpH, in addition to Δψ, contributes energetically to Tat transport in vivo in E. coli spheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway.

  PublisherOA PDFDOI

• Cell-penetrating peptides stimulate protein transport on the Twin-arginine translocation pathway

  bioRxiv (Cold Spring Harbor Laboratory) · 2023-07-09 · 3 citations

  preprintOpen accessSenior authorCorresponding

  Abstract The Tat pathway is essential for photosynthetic protein transport across plant thylakoid membranes and is also ubiquitous throughout prokaryotes and archaea. The Tat pathway is unique amongst protein translocation pathways as it specializes in transporting folded proteins driven by a proton motive force. Mechanistic details of the actual translocation step (s) of the pathway remain elusive. Here, we show that membrane thinning stimulates Tat transport and, conversely, membrane strengthening abolishes Tat transport. We draw parallels from the Tat transport mechanism to that of cell penetrating peptides and propose that the Tat pore could be toroidal in shape and lined by lipids, as in those formed by cell penetrating peptides. Significance Statement Protein translocation across membranes is a significant cellular activity in both prokaryotes and eukaryotes. The Tat pathway for protein translocation operates in bacteria, archaea, chloroplasts, and plant mitochondria. Its mechanism of action has been difficult to decipher, but recent evidence suggests it does not use a conical proteinaceous transport channel. Instead, it has been suggested to translocate proteins through lipid-lined toroidal pores set up by membrane thinning. This work supports that hypothesis by showing that membrane-thinning cell-penetrating peptides stimulate the Tat pathway in both chloroplasts and bacterial plasma membranes, and that membrane stabilization blocks the pathway. We believe this is the most direct evidence to date of the toroidal pore mechanism operating in the Tat pathway.

  PublisherOA PDFDOI

• Characterization of thylakoid division using chloroplast dividing mutants in Arabidopsis

  Photosynthesis Research · 2023-03-01 · 2 citations

  articleOpen accessSenior author
  PublisherOA PDFDOI

• The polar amino acid in the TatA transmembrane helix is not strictly necessary for protein function

  Journal of Biological Chemistry · 2023-02-09 · 4 citations

  articleOpen accessSenior authorCorresponding

  The twin-arginine translocation (Tat) pathway utilizes the proton-motive force to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, we examined a complete set of 19-amino-acid substitutions. Unexpectedly, although the polar amino acid is preferred overall, our experiments suggest that it is not necessary for a functional TatA. Hydrophilicity and helix-stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophilicity, which was demonstrated to impact the Tat transport activity. Furthermore, we identified a four-residue motif at the N terminus of the TatA TMH by sequence alignment. Using a biochemical approach, we found that the N-terminal motif was functionally significant, with evidence indicating a potential role in the preference for utilizing different proton-motive force components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism. The twin-arginine translocation (Tat) pathway utilizes the proton-motive force to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, we examined a complete set of 19-amino-acid substitutions. Unexpectedly, although the polar amino acid is preferred overall, our experiments suggest that it is not necessary for a functional TatA. Hydrophilicity and helix-stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophilicity, which was demonstrated to impact the Tat transport activity. Furthermore, we identified a four-residue motif at the N terminus of the TatA TMH by sequence alignment. Using a biochemical approach, we found that the N-terminal motif was functionally significant, with evidence indicating a potential role in the preference for utilizing different proton-motive force components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism. The twin-arginine translocation (Tat) pathway, a protein transport machinery, is found in bacteria, archaebacteria, chloroplasts, and plant mitochondria. In bacteria, the Tat pathway is involved in many critical biological processes, including cell division, stress tolerance, and electron transport (1Berks B.C. The twin-arginine protein translocation pathway.Annu. Rev. Biochem. 2015; 84: 843-864Crossref PubMed Scopus (110) Google Scholar, 2Gohlke U. Pullan L. McDevitt C.A. Porcelli I. Leeuw E de Palmer T. et al.The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10482-10486Crossref PubMed Scopus (220) Google Scholar, 3Ize B. Stanley N.R. Buchanan G. Palmer T. Role of the Escherichia coli Tat pathway in outer membrane integrity.Mol. Microbiol. 2003; 48: 1183-1193Crossref PubMed Scopus (171) Google Scholar). In plants, the Tat pathway transports several proteins that are essential for photosynthesis across thylakoid membranes (4Clark S.A. Theg S.M. A folded protein can be transported across the chloroplast envelope and thylakoid membranes.Mol. Biol. Cell. 1997; 8: 923-934Crossref PubMed Scopus (147) Google Scholar). In haloarchaea, which normally possess high cytoplasmic salt concentrations leading to faster protein folding, the Tat pathway serves almost 50% of secretome (5Ghosh D. Boral D. Vankudoth K.R. Ramasamy S. Analysis of haloarchaeal twin-arginine translocase pathway reveals the diversity of the machineries.Heliyon. 2019; 5e01587Abstract Full Text Full Text PDF Scopus (3) Google Scholar). Unlike the similarly ubiquitous Sec pathway, the Tat pathway is able to transport folded proteins (4Clark S.A. Theg S.M. A folded protein can be transported across the chloroplast envelope and thylakoid membranes.Mol. Biol. Cell. 1997; 8: 923-934Crossref PubMed Scopus (147) Google Scholar) while requiring only the proton-motive force (pmf), with no contribution from NTP hydrolysis (6Braun N.A. Davis A.W. Theg S.M. The chloroplast Tat pathway utilizes the transmembrane electric potential as Full Text Full Text PDF PubMed Scopus Google Scholar). The Tat system in most of three subunits, TatA, TatB, and (1Berks B.C. The twin-arginine protein translocation pathway.Annu. Rev. Biochem. 2015; 84: 843-864Crossref PubMed Scopus (110) Google Scholar). shown that three can a which as a for of Tat the to the Tat TatA are to the and are present in a in the TatA is present at to T. Buchanan G. S. et of the TatA component of the twin-arginine protein transport system by in Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, a role in thylakoid protein PubMed Scopus Google Scholar, U. Leeuw E de Stanley N.R. Palmer T. et components of the Escherichia coli Tat protein transport system a Biochem. PubMed Scopus Google is TatA protein PubMed Scopus Google Scholar). The of the three Tat have B. of the TatA component of the twin-arginine protein transport system from PubMed Scopus Google Scholar, L. and of plant TatA in and by PubMed Scopus Google Scholar, S. et of the of the twin-arginine protein transport PubMed Scopus Google Scholar, L. of the component of the twin-arginine translocation PubMed Scopus Google Scholar). TatA and possess a transmembrane helix (TMH) by a and a and a the Tat transports folded proteins of different Theg S.M. shift the membrane of transport and Sec Natl. Acad. Sci. U. S. A. Scopus Google Scholar, S.A. Theg S.M. thylakoid membranes highly to protein Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). that this in the of a the Sec A. A. S. the Sec Rev. Microbiol. PubMed Scopus Google Scholar). that the Tat pathway by in utilizing a Theg S.M. shift the membrane of transport and Sec Natl. Acad. Sci. U. S. A. Scopus Google Scholar, T. of the translocation 2003; PubMed Scopus Google Scholar, B. Theg S.M. is a in protein transport across membranes the Tat Biol. Full Text Full Text PDF Scopus (3) Google Scholar). Previous studies have demonstrated that have a highly TMH B. Theg S.M. is a in protein transport across membranes the Tat Biol. Full Text Full Text PDF Scopus (3) Google which membrane and is critical to the Tat B. Theg S.M. is a in protein transport across membranes the Tat Biol. Full Text Full Text PDF Scopus (3) Google Scholar, B. D. T. The TatA component of the twin-arginine translocation system the cytoplasmic membrane of Escherichia coli protein Biol. 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  PublisherOA PDFDOI

• A Real-Time Analysis of Protein Transport via the Twin Arginine Translocation Pathway in Response to Different Components of the Protonmotive Force <i>in vivo</i>

  bioRxiv (Cold Spring Harbor Laboratory) · 2023-01-13 · 1 citations

  preprintOpen accessSenior authorCorresponding

  Abstract The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the Δψ component of PMF in vitro . To investigate the comprehensive PMF requirement in Escherichia coli , we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology (NanoBiT) in E. coli spheroplasts. This luminescence assay allows for continuous monitoring of Tat transport with high-resolution, making it possible to observe subtle changes in transport in response to different treatments. By applying the NanoLuc assay, we report that, under acidic conditions, ΔpH, in addition to Δψ, contributes energetically to Tat transport in vivo in E. coli spheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway.

  PublisherOA PDFDOI

• Hydrophobic mismatch is a key factor in protein transport across lipid bilayer membranes via the Tat pathway

  Journal of Biological Chemistry · 2022-04-28 · 20 citations

  articleOpen accessSenior authorCorresponding

  The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoids, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits: TatA, TatB, and TatC. TatA and TatB possess short transmembrane alpha helices (TMHs), both of which are only 15 residues long in Escherichia coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer, although the functional significance of this mismatch is unclear. Here, we sought to address the functional importance of the hydrophobic mismatch in the Tat transport mechanism in E. coli. We conducted three different assays to evaluate the effect of TMH length mutants on Tat activity and observed that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off following any modification of this length. Surprisingly, TatA and TatB with as few as 11 residues in their TMHs can still insert into the membrane bilayer, albeit with a decline in membrane integrity. These findings support a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, we conclude that the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes.

  PublisherOA PDFDOI

Recent grants

• Functions of Stromal Chaperones in Chloroplasts of Physcomitrella Patens

  NSF · $671k · 2010–2013

• Probing the Interaction of Precursors with the Chloroplast Import Machinery

  NSF · $300k · 2013–2016

• Chloroplast Protein Transport in the Moss Physcomitrella Patens

  NSF · $488k · 2003–2007

Frequent coauthors

• Yasuo Ishikawa

  Okayama University

  10 shared

• Noriaki Tamura

  Fukuoka Women's University

  10 shared

• Yasusi Yamamoto

  Okayama University

  10 shared

• Mayuko Otsubo

  Fukuoka Women's University

  10 shared

• Sabeeha Merchant

  9 shared

• Iniyan Ganesan

  University of Freiburg

  8 shared

• Lan-Xin Shi

  University of California, Davis

  8 shared

• Nikolai A. Braun

  University of Manchester

  7 shared

Awards & honors

• AAAS Names Three College of Biological Sciences Researchers…