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Miami University
1.
Ma, Qianqian.
FUNCTIONAL INTERACTION ANALYSIS OF CHLOROPLAST TWIN ARGININE
TRANSLOCATION (CPTAT) PATHWAY AND ITS ROLE IN PLASTID
DEVELOPMENT.
Degree: PhD, Cell, Molecular and Structural Biology
(CMSB), 2016, Miami University
URL: http://rave.ohiolink.edu/etdc/view?acc_num=miami1477671264917956 
The Twin arginine translocation (Tat) system is an
ancient and evolutionarily conserved system which translocates
fully-folded proteins across thylakoidal membrane in chloroplasts
as well as cytoplasmic membrane of eubacteria and archaebacteria.
In chloroplasts, the cpTat system carries out the function in
translocating roughly half of the critical photosynthetic lumenal
proteins. During the past two decades, the core components of this
protein translocase has been identified, and the representative
structures of the components from bacterial homologues have been
obtained. What still remains largely unknown is the molecular
mechanism of the cpTat pathway. Understanding how cpTat components
interact will provide important clues towards solving the transport
mechanism of cpTat. Two core components, Hcf106 and cpTatC, play a
major role in binding the substrate signal peptide and recruiting
of the pore-forming component Tha4. This dissertation mainly
describes the development of a technique using thylakoid-integrated
recombinant Hcf106 to study its contribution to the cpTat pathway
and usage of the recombinant Hcf106 as a probe to investigate the
interaction between Hcf106 and other cpTat components. We detected
close contacts between Hcf106 and cpTatC and mapped the
architecture of the Hcf106-cpTatC complex. In addition, we trapped
a Hcf106-cpTatC-Tha4 trimer via their transmembrane helices for the
first time. All these data support a model that Hcf106 plays a role
in controlling Tha4 entering the cavity of cpTatC, which initiates
precursor translocation.Most of the knowledge concerning the
mechanism of cpTat pathway in chloroplasts has been obtained
through experiments with isolated pea chloroplasts, and it is
assumed that all higher plants utilize the same general transport
apparatus. To further understand the role of cpTat in plastid
development, we characterized a HCF106 mutant of cpTat pathway,
pgw, in the model plant Arabidopsis thaliana to investigate how
cpTat system deficiency affects the thylakoids biogenesis and plant
development. We detected underdeveloped thylakoids and defects in
cpTat transport in pgw, and a possible retrograde signaling pathway
was associated with the cpTat deficiency. We conclude that as one
of the core component of cpTat pathway, HCF106 plays an important
role in Arabidopsis thylakoid development.
Advisors/Committee Members: Dabney-Smith, Carole (Advisor), Crowder, Michael (Committee Chair).Subjects/Keywords: Biology; Biochemistry; Cellular Biology; chloroplast twin-arginine translocation; cpTat; Hcf106; thylakoid; cross-linking; Arabidopsis
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APA (6th Edition):
Ma, Q. (2016). FUNCTIONAL INTERACTION ANALYSIS OF CHLOROPLAST TWIN ARGININE
TRANSLOCATION (CPTAT) PATHWAY AND ITS ROLE IN PLASTID
DEVELOPMENT. (Doctoral Dissertation). Miami University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=miami1477671264917956
Chicago Manual of Style (16th Edition):
Ma, Qianqian. “FUNCTIONAL INTERACTION ANALYSIS OF CHLOROPLAST TWIN ARGININE
TRANSLOCATION (CPTAT) PATHWAY AND ITS ROLE IN PLASTID
DEVELOPMENT.” 2016. Doctoral Dissertation, Miami University. Accessed January 18, 2021.
http://rave.ohiolink.edu/etdc/view?acc_num=miami1477671264917956.
MLA Handbook (7th Edition):
Ma, Qianqian. “FUNCTIONAL INTERACTION ANALYSIS OF CHLOROPLAST TWIN ARGININE
TRANSLOCATION (CPTAT) PATHWAY AND ITS ROLE IN PLASTID
DEVELOPMENT.” 2016. Web. 18 Jan 2021.
Vancouver:
Ma Q. FUNCTIONAL INTERACTION ANALYSIS OF CHLOROPLAST TWIN ARGININE
TRANSLOCATION (CPTAT) PATHWAY AND ITS ROLE IN PLASTID
DEVELOPMENT. [Internet] [Doctoral dissertation]. Miami University; 2016. [cited 2021 Jan 18].
Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1477671264917956.
Council of Science Editors:
Ma Q. FUNCTIONAL INTERACTION ANALYSIS OF CHLOROPLAST TWIN ARGININE
TRANSLOCATION (CPTAT) PATHWAY AND ITS ROLE IN PLASTID
DEVELOPMENT. [Doctoral Dissertation]. Miami University; 2016. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1477671264917956
Miami University
2.
New, Christopher Paul.
Analysis of Tha4 Function and Organization in Chloroplast
Twin Arginine Transport.
Degree: PhD, Cell, Molecular and Structural Biology
(CMSB), 2020, Miami University
URL: http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538
The chloroplast Twin Arginine Translocase (cpTAT)
system transports fully folded proteins across the thylakoid
membrane in plant cells using only energy derived from the proton
motive force (PMF). Three membrane bound component proteins:
cpTatC, Hcf106, and Tha4 function together in a transient manner to
accomplish transport. However, clear mechanistic details of this
process remain elusive such as how cpTAT utilizes energy stored in
the PMF or how the individual component proteins interact during
each step of transport. In addition, prior structural
characterization of (cp)TAT proteins used truncated versions of the
components. This dissertation describes work to develop methods to
purify full-length Hcf106 for biophysical characterization.
Additionally, this dissertation details the work to determine the
function of a membrane embedded glutamate in the Tha4 transmembrane
helix (TMH).A series of purification trials were carried out to
isolate Hcf106 fused to maltose binding protein (MBP) by the
recognition sequence of tobacco etch virus protease (TEVp). Fusion
protein and protease were expressed in and purified from E. coli
using affinity chromatography. Multiple parameters and additives
were tested during optimization of TEVp proteolysis reactions with
MBP-Hcf106. TEVp and free MBP were separated from un-cleaved
MBP-Hcf106 and free Hcf106 by affinity and size exclusion
chromatography. Although TEVp and free MBP were removed after an
optimized proteolysis reaction, free Hcf106 showed its recalcitrant
nature through resistance of separation from un-cleaved MBP-Hcf106
by size exclusion chromatography in several detergent and buffer
conditions.To better understand the role of the membrane embedded
Tha4 glutamate 10 (E10), Tha4 variants with glutamate to alanine
(E10A) or glutamate to aspartate (E10D) substitutions were used to
complement loss of cpTAT function in thylakoid membranes.
Sequential glutamate substitutions in the TMH of Tha4 variant E10A
were unable to restore transport while aspartate substitutions were
mildly able to complement loss of function. Furthermore,
organization between three structural regions in Tha4 E10/A/D
variants was determined by disulfide crosslinking during various
transport conditions. Tha4 E10/A/D variant oligomer formation was
enhanced in the presence of functional precursor with and without
PMF present. An increase in TMH hydrophobicity by alanine
substitution was shown to increase Tha4 stability in isolated
thylakoid membranes and to promote tighter packing interactions
between adjacent Tha4 monomers. The interaction data was then used
to develop a model of how Tha4 E10/A/D variant tetramers pack and
reorganize in the presence of precursor.
Advisors/Committee Members: Dabney-Smith, Carole (Advisor), Page, Rick (Committee Chair).Subjects/Keywords: Biochemistry; Cellular Biology; Plant Biology; Molecular Biology; chloroplast twin arginine transport; protein transport; cpTAT; TAT; Tha4; Hcf106; protein purification; maltose binding protein affinity chromatography; oligomer formation; complementation; transmembrane domain hydrophobicity
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APA (6th Edition):
New, C. P. (2020). Analysis of Tha4 Function and Organization in Chloroplast
Twin Arginine Transport. (Doctoral Dissertation). Miami University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538
Chicago Manual of Style (16th Edition):
New, Christopher Paul. “Analysis of Tha4 Function and Organization in Chloroplast
Twin Arginine Transport.” 2020. Doctoral Dissertation, Miami University. Accessed January 18, 2021.
http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538.
MLA Handbook (7th Edition):
New, Christopher Paul. “Analysis of Tha4 Function and Organization in Chloroplast
Twin Arginine Transport.” 2020. Web. 18 Jan 2021.
Vancouver:
New CP. Analysis of Tha4 Function and Organization in Chloroplast
Twin Arginine Transport. [Internet] [Doctoral dissertation]. Miami University; 2020. [cited 2021 Jan 18].
Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538.
Council of Science Editors:
New CP. Analysis of Tha4 Function and Organization in Chloroplast
Twin Arginine Transport. [Doctoral Dissertation]. Miami University; 2020. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538
3.
Storm, Amanda R.
Structural studies of cpTat component Tha4 in both native
and synthetic membrane systems.
Degree: PhD, Chemistry, 2013, Miami University
URL: http://rave.ohiolink.edu/etdc/view?acc_num=miami1385466330
The chloroplast Twin Arginine Translocation (cpTat)
system facilitates transport of about half of all proteins that
cross the thylakoid membrane in plant chloroplasts. Known
mechanistic features of the cpTat system are drastically different
from other known translocation systems, notably in its formation of
a transient complex to transport fully folded proteins utilizing
only the proton-motive force for energy. Tha4, one of the
transmembrane cpTat components, is proposed to function as the
primary pore component by potentially creating a weak 'patch' in
the membrane or undergoing a conformational change to facilitate
transport. Yet, there is limited knowledge about the basic topology
or structure of Tha4 and little work has been done to detect any
transport-stimulated conformational changes. This dissertation
describes the development and use of biochemical and biophysical
techniques to explore aspects of Tha4 structure in both native and
synthetic membrane systems.Tha4 topology in isolated thylakoid
membranes was determined under both resting state and transport
conditions. A modified substituted cysteine accessibility method
(SCAM) indicated that Tha4 adopts an N-in, C-out topology with a
single transmembrane domain (TMD) and a tilted amphipathic helix
(APH). Cysteine and protease accessibility assays performed under
transport-stimulating conditions detected significant changes in
the accessibility of the C-terminal APH region of Tha4 but no
global changes in the over-all topology.In preparation for more
detailed biophysical structural studies, procedures were developed
and optimized for the over-expression and purification of Tha4 and
insertion of spin-labeled protein into four different lipid
systems, including a novel lipodisq nanoparticle architecture.
Topology of integrated spin labeled Tha4 detected by electron
paramagnetic resonance (EPR) spectroscopy was in agreement with
topology observed in thylakoids. Tha4 lipodisq nanoparticles
containing native thylakoid lipids were also generated and purified
by nickel affinity chromatography. An additional project describes
the development of methods for incorporation of the rigid TOAC
(2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid)
spin label into full length proteins using amber codon suppression
and in vitro translation. Continuous-exchange cell-free translation
techniques were used to improve yield of TOAC-labeled protein and
co-translationally insert protein into liposomes. Issues with
inefficient reagent synthesis hindered the large scale use of these
methods.
Advisors/Committee Members: Dabney-Smith, Carole (Advisor), Lorigan, Gary (Committee Chair).Subjects/Keywords: Biochemistry; Chemistry; chloroplast twin arginine translocation; cpTat; Tha4; thylakoid; native topology; electron paramagnetic resonance; EPR; lipodisq nanoparticles; TOAC; amber suppression
…7
Figure 1.3: Protein translocation by the cpTat pathway… …61
Figure 3.4: Recombinant Tha4 restores cpTat transport… …cpTat (19–21). The
4
first two pathways are strictly involved in integration of… …the two major protein translocases of the thylakoid membrane are the
cpSec and cpTat… …functionality in the E. coli Sec translocase
(81).
1.2.4 cpTat pathway
1.2.4.1 Introduction…
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APA ·
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MLA ·
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CSE |
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Manager
APA (6th Edition):
Storm, A. R. (2013). Structural studies of cpTat component Tha4 in both native
and synthetic membrane systems. (Doctoral Dissertation). Miami University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=miami1385466330
Chicago Manual of Style (16th Edition):
Storm, Amanda R. “Structural studies of cpTat component Tha4 in both native
and synthetic membrane systems.” 2013. Doctoral Dissertation, Miami University. Accessed January 18, 2021.
http://rave.ohiolink.edu/etdc/view?acc_num=miami1385466330.
MLA Handbook (7th Edition):
Storm, Amanda R. “Structural studies of cpTat component Tha4 in both native
and synthetic membrane systems.” 2013. Web. 18 Jan 2021.
Vancouver:
Storm AR. Structural studies of cpTat component Tha4 in both native
and synthetic membrane systems. [Internet] [Doctoral dissertation]. Miami University; 2013. [cited 2021 Jan 18].
Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1385466330.
Council of Science Editors:
Storm AR. Structural studies of cpTat component Tha4 in both native
and synthetic membrane systems. [Doctoral Dissertation]. Miami University; 2013. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1385466330
. 
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