ABSTRACT

INVESTIGATING THE PORE COMPOSITION OF THE CHLOROPLAST TWIN ARGININE TRANSPORT SYSTEM

by Nefertiti Muhammad

The cpTat system transports fully folded proteins into the thylakoid using the proton motive force and, together, cpTatC, Tha4, and Hcf106 transport precursors across the bilayer membrane. Tha4 is predicted to form the translocation pore; however, Hcf106 may also have a role because of the large size of some cpTat substrates and its homology to Tha4. In this study, direct binding between Hcf106 and a cpTat substrate, the Oxygen Evolving Complex precursor (pOE17), was investigated. Prior work showed direct binding to Tha4 for four variants of the substrate (pOE17-D68C, -S84C, -K99C, and -T115C) using cysteine crosslinking. The current study showed little to no direct binding between Hcf106 and the last variant of the substrate (pOE17-T115C); although, that variant could interact with Tha4. Together with precursor-Tha4 interaction maps, we concluded that both cpTat components bind directly to the substrate and the strongest interactions occur at the C-tail and the C-terminal end of the amphipathic helix (APH) region. This study also investigates whether cpTat substrates stay in contact with Hcf106 throughout transport. Protease treatments were performed on crosslinked Hcf106 and pOE17 at the C-tail and N-terminal residues of Hcf106. Unexpectedly, membrane-protected and - unprotected crosslinked products were detected in the N-terminus of Hcf106. The paper presents limitations with the study and directions for future research.

INVESTIGATING THE PORE COMPOSITION OF THE CHLOROPLAST TWIN ARGININE TRANSPORT SYSTEM

A THESIS

Submitted to the Faculty of Miami University in partial fulfillment of the requirements of the degree of Master of Science Cell, Molecular, and Structural Biology Program by Nefertiti Muhammad Miami University Oxford, Ohio 2018

Carole Dabney-Smith, Ph.D., Advisor

Gary A. Lorigan, Ph.D., Reader

Anne E. Hagerman, Ph.D., Reader

This Thesis titled

INVESTIGATING THE PORE COMPOSITION OF THE CHLOROPLAST TWIN ARGININE TRANSPORT SYSTEM

by Nefertiti Muhammad

has been approved for publication by

The College of Arts and Science and The Cell, Molecular, and Structural Biology Program

______Carole Dabney-Smith, Ph. D.

______Gary A. Lorigan, Ph. D.

______Ann E. Hagerman, Ph. D.

©

Nefertiti Muhammad 2018

Table of Contents CHAPTER 1 Introduction ...... 1 1.1 Overview of Chloroplast Transporter Proteins ...... 2 1.2 Chaperones and Regulatory Proteins ...... 4 1.3 TOC ...... 5 1.31 TOC Components ...... 5 1.32 The TOC Model ...... 8 1.4 TIC ...... 8 1.41 TIC Components ...... 8 1.42 The TIC Model...... 11 1.5 SRP ...... 11 1.6 Secretory System...... 13 1.7 Twin Arginine Translocation System ...... 14 1.8 Thesis Summary ...... 17 1.9 References ...... 17 CHAPTER 2 INVESTIGATING THE PORE COMPOSITION OF THE CHLOROPLAST TWIN ARGININE TRANSPORT SYSTEM ...... 36 2.1 Abstract ...... 37 2.2 Introduction ...... 37 2.3 Results ...... 39 2.31 Oxygen Evolving Complex Crosslinked Hcf106 at the C-Proximal APH and C-tail ..39 2.32 Hcf16 Crosslinked to The Precursor and Mature Protein of OE17-T115C Under Transport Conditions ...... 42 2.4 Discussion ...... 42 2.41 Hcf106 Integrated Interaction Map...... 42 2.42 Combination of Crosslinked & Un-crosslinked OE17T115C Recovered ...... 44 2.43 cpTat Transport Model: Membrane Weakening ...... 45 2.5 Methods ...... 47 2.51 IN VITRO SYNTHESIS OF HCF106 AND TAT SUBSTRATE CONSTRUCTS ...... 47 2.52 ISOLATION OF CHLOROPLASTS AND THYLAKOIDS ...... 47 2.53 PRECURSOR RADIOLABEL-INCORPORATED TRANSLATION ...... 47 2.54 PRECURSOR INCUBATION WITH DTNB ...... 47 2.55 HCF106 INTEGRATION INTO THYLAKOIDS ...... 48 2.56 TRANSPORT ASSAY ...... 48 2.57 THERMOLYSIN TREATMENT ...... 48 iii

2.58 MEMBRANE SOLUBILIZATION ...... 48 2.59 NICKEL-AFFINITY PULL-DOWN ...... 48 2.510 CROSSLINK-PRODUCT DETECTION ...... 49 2.6 References ...... 49 CHAPTER 3 Conclusions ...... 52 3.1 Conclusion ...... 53 3.2 References ...... 55

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List of Tables Table 2.1 INTEGRATED TABLE OF CROSSLINKING BETWEEN POE17 AND HCF106 VARIANTS ...... 41

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List of Figures Figure 1.1 THE STRUCTURE AND FUNCTION OF CHLOROPLAST AND THYLAKOIDS ...... 2 Figure 1.2 A MODEL OF TOC AND TIC...... 10 Figure 1.3 A MECHANISTIC MODEL OF THE CHLOROPLAST TWIN ARGININE TRANSLOCATION (CPTAT) PATHWAY...... 16 Figure 2.1 VARIANTS OF OXYGEN EVOLVING COMPLEX AND HCF106 ...... 39 Figure 2.2 HCF106 CROSSLINKING FOR OE17-D68C AND –T115C ...... 40 Figure 2.3 CROSSLINKING AND THERMOLYSIN ASSAY FOR OE17-T115C AND HCF106-G6C AND –I64C .....42 Figure 2.4 CHLOROPLAST TWIN ARGININE TRANSLOCATION PATHWAY MEMBRANE WEAKENING TRANSPORT MODEL ...... 46

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Dedication For Calvin Muhammad “I loved talking with you, I miss you everyday.” -Much love from your daughter, Nefertiti

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Acknowledgments This Master’s thesis would not have been possible without the support of my graduate advisor, Dr. Carole Dabney-Smith. I am deeply thankful for her mentorship during my time in the Miami University REU program, leading me to go beyond my undergraduate education. I was more than fortunate to have her as my graduate research advisor in the Cell, Molecular, and Structural Biology program. It’s not often for graduate students to have an advisor so invested in their development as a researcher and their future after graduate school. I especially appreciate her support when I returned home for medical reasons. I could not have completed my thesis work without her patience and compassion. I feel very lucky to have worked in Dr. Dabney-Smih’s lab. In addition to her, I would like to acknowledge other faculty members, Dr. Lorigan, and Dr. Ellen J. Yezierski for their support during my time at Miami University. Tremendous amount of thanks to my lab mates, Dr. Debjani Pal, Dr. Amanda Storm, Dr. Qianqian Ma, Dr. Lei Zhang, Paul New, and Ramja Sritharan, and our lab technician, Martin Smith. I was lucky to join an amazing cohort who helped me learn the basics of the lab,who gave me critical feedback on my work and helped me work through tough parts of my research. I am also very grateful to Dr. Lorigan for welcoming me into his lab during my first year at Miami University. In addition, Kaylee Roy Gibson, Dr. Indra D. Sahu, and Dr. Rongfu Zhang were a great help when I was getting acclimated to the lab and learning about my first research project. Lastly, I would like to thank my family. To my mother, Judith Muhammad, I don’t think it’s humanly possible to repay you for all of your sacrifices. I am so appreciative of the time and energy you sacrificed to get me to this point. I would not have made it had it not been for your constant support and love. To my father, Calvin Muhammad, you were confident in me and always reminded me what I was capable of accomplishing. Thank you so much for that. I love you and miss you dearly. To my grandmother, Edith Stapleton, and my cousin, Marie Nicole Johnson-Watts, I specifically remember you talking me through some rough patches. Your strong words of encouragement and compassion got me back on track and uplifted my spirit. I would also like to thank Dr. Joe Nee from Miami University Student Counseling Services. He went above and beyond his duty as a counselor and helped me through my last year at Miami University. Before I close, I have to extend a warm thank you to the Honorable Minister Louis Farrakhan of the Nation of Islam and everyone at Faith Baptist Church for being a source of encouragement and community while I was away from home. From the pulpit, Minister Farrakhan instilled in me and my peers a great love of the hard sciences. He had high expectations for us and he is the reason why I chose to study biochemistry. From Faith Baptist Church, I appreciate Dr. Whitney Craig and Dr. Andrew Craig for going out of their way to pick me up every Sunday for Church. Pastor Eric, his wife Tonya, Deacon Donald and his wife Jane Dawley were so warm and kind for welcoming me into their homes for the holidays and bible study. I am so thankful I had an opportunity to be a part of such a wonderful community. I had a wonderful experience at Miami University, and I could not have made it to the finish line without all of these wonderful people.

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Chapter 1. INTRODUCTION

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1.1 OVERVIEW OF CHLOROPLAST TRANSPORTER PROTEINS In plants and algae, chloroplasts are the organelles where photosynthesis, amino acid, fatty acid and terpene synthesis take place (Shi and Theg 2013). There are three membranes and three aqueous compartments where these metabolic processes can occur: the outer envelope, the inner envelope, the thylakoid membrane, the inner membrane space (IMS), the stroma or the thylakoid lumen (Figure 1.1). For example, photosynthesis light-dependent reactions occur on the thylakoid membrane while the light-independent reactions occur in the stroma. During chloroplast biogenesis, all of the photosynthetic enzymes and chaperones are sorted to their proper compartments. Compartmentalization is accomplished by targeting proteins to transport systems that can move them across lipid bilayers. There are ~3,000 chloroplast proteins (Leister 2003). The majority of them (~95%) are encoded by the nucleus and the remainder are encoded by the chloroplast genome (Shi and Theg 2013) as a result of an endosymbiotic event, where cyanobacteria were engulfed by a eukaryote, ~1.5 billion years ago (Yoon et al. 2004). Over time, the endosymbiont relinquished the majority of its genome to the host. Consequently, protein synthesis in the chloroplast is coordinated between the nucleus and the chloroplast. Proteins encoded by the chloroplast genome are synthesized in the stroma by the chloroplast ribosome. The chloroplast ribosome is a bacterial-type 70S ribosome (Tiller and Block 2014), composed of a 50S and 30S subunit. Nuclear-encoded chloroplast proteins are synthesized in the cytosol by cytosolic ribosomes. The cytosolic ribosomes are eukaryotic 80S ribosomes, composed of a 40S and 60S subunit (Giavalisco 2005).

Figure 1.1 THE STRUCTURE AND FUNCTION OF CHLOROPLAST AND THYLAKOIDS

The chloroplast is composed of three aqueous compartments and three membrane bilayers. The outer and inner envelope membranes enclose the aqueous intermembrane space. The aqueous stroma is enclosed by the inner envelope membrane and contains granum (stacks of thylakoids) connected by stroma lamella structures. The thylakoid is composed of a thylakoid membrane that encloses an aqueous lumen (adapted from TutorVista.com, 2018). 2

Unlike plastid-encoded proteins, nuclear-encoded proteins required the development of targeting sequences on the N-terminus, transit peptides, to be sorted from the cytoplasm to the chloroplast. In addition, transport machinery was developed from both the host and endosymbiont to transport the nuclear-encoded proteins. Chloroplasts have transport systems on each the outer and inner envelope, which serve to bring in or import cytosolic precursor proteins, and three transport systems on the thylakoid membrane. The transport systems are composed of membrane proteins and chaperone proteins, often requiring nucleotide hydrolysis for energy. Most transporters accommodate unfolded and partially unfolded substrates and chaperones play roles in unfolding/refolding substrates at the start and end of transport in an ATP-dependent manner. Nuclear- and plastid-encoded proteins are targeted to the correct transport system with targeting sequences, which are N-terminal amino acid extensions on protein substrates that recognize and bind to receptor proteins. Transit peptide sequences are found on precursors synthesized in the cytoplasm, are not conserved and vary widely in length (Bruce 2001; Bruce 2000); although, most do contain similar physicochemical properties such as having a loose random coil structure, containing binding sites for chaperones, being slightly basic with an arginine-enriched C-terminus, followed by a domain with an abundance of serine residues, and an uncharged N-terminal domain (Claros et al. 1997; Jarvis 2008; Bruce 2001; Bruce 2000). Transit peptides contain targeting and routing information to direct chloroplast precursors to their correct sub-organellar location, meaning they may contain targeting information directing precursors across multiple membranes. For example, nuclear-encoded proteins destined to the thylakoid lumen require a bipartite targeting sequence that directs them across the outer and inner envelopes as well as the thylakoid membrane. In contrast, plastid-encoded proteins destined to the thylakoid require a targeting sequence called a signal peptide, which is similar to prokaryotic signal peptides. Following transport, the targeting sequence is removed by proteolytic cleavage, revealing a fully mature protein if the protein is at its final destination. There are two transport systems on the outer and inner envelope in the chloroplast, and three novel and ancient transport systems on the thylakoid membrane (Schnell et al. 1997). TOC (Translocon at the Outer Chloroplast Envelope Membrane) and TIC (Translocon at the Inner Chloroplast Envelope Membrane) work in tandem to import substrates from the cytosol into the chloroplast (Figure 1.2). Heat shock proteins and other chaperones as well as ATPases guide the precursor and harness energy for transport. Stromal processing peptidase (SPP) removes the transit peptide from the precursor substrates after transport releasing the mature protein or exposing a second targeting sequence for subsequent routing. There are four main pathways to route these intermediate proteins to the thylakoid: spontaneous insertion, the SRP (Signal Recognition Particle), Sec (General Secretory) and cpTat (chloroplast Twin Arginine Translocation) pathway (Schleiff 2001; Cline 2008; Aldridge 2009; Celedon 2012). In spontaneous insertion, substrates are integrated into the membrane bilayer without the assistance of transport proteins or chaperones. The SRP pathway imports membrane proteins from the Light-Harvesting Chlorophyll Binding Protein (LHBP) family. The Sec and Tat pathway are highly conserved systems that can work separately or in parallel to import proteins across the thylakoid membrane. The cpTat system transports proteins involved in photosynthesis into the lumen, and in bacteria it exports proteins involved in, among other processes, anaerobic metabolism. The Sec system transports partially unfolded proteins and the Tat system is able to import fully folded proteins. The Tat and Sec pathways also differ in terms of energy; the Tat system is powered by the proton

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motive force (PMF) generated from respiration or the light-dependent reactions while the Sec system relies mostly on nucleotide hydrolysis; although the PMF enhances Sec transport. A review of chloroplast transport systems is relevant to the study presented in chapter two, which focuses on the cpTat transport mechanism. Unlike other chloroplast transport systems, the cpTat system transports fully folded proteins of various sizes. In addition, it does this without disrupting the integrity of the membrane or dissipating the PMF. Furthermore, the cpTat system is coupled to the PMF, rather than nucleotide hydrolysis, to drive transport across the membrane. The structure of the pore, how the membrane and PMF are maintained, and how the PMF is coupled to transport is still being investigated. The rest of this chapter will provide the context leading up to the chapter two, which probes the composition of the cpTat pore.

1.2 CHAPERONES AND REGULATORY PROTEINS Targeting nuclear-encoded proteins to the chloroplast involves substrate proofreading, (un)folding, and stabilization by chaperone and regulatory proteins. A number of heat-shock proteins (Hsp70, Hsp93 and Hsp90) were shown to be involved in chloroplast import (Fellerer 2011; Huang 2015; Chotewutmontri 2015). Heat-shock proteins are a family of proteins that are expressed in response to stress. They are responsible for assembling multi-protein complexes, (un)folding and sorting proteins, and performing cell cycle-control (Hartl 2011). The 70 kDa heat-shock protein (Hsp70), was shown to directly interact with precursor substrates (Rial et al. 2000; May and Soll 2000). Chotewutmontri and Bruce demonstrated that most transit peptides (TP) use H70BS to initiate transport in a study by substituting the N-terminal domain of the TP of the small subunit of Rubisco with a strong Hsp70 recognition element (H70BS). Also, plastid Hsp70 functions as a translocation motor in the stroma, pulling substrates across the bilayer (Su and Li 2010). Direct binding between cytosolic and plastid Hsp70 and the transit peptide of ferredoxin-NADP+ reductase precursor (pre-FNRtp) was shown in a binding assay (Rial et al. 2000). Despite Hsp70’s involvement, another study suggested that strong interactions between stromal Hsp70 and the transit peptide may not be necessary for import (Rial et al. 2003). pre-FNRtp with low affinity for Hsp70 was imported with the same apparent KM as the wild type and a two-fold increase in Vmax of wild type. Rial et al. speculate that multiple chaperones and regulatory proteins are involved in targeting substrates to the stroma. In another study, cytosolic Hsp70 and another chaperone, 14-3-3, formed a complex with precursors to facilitate initial interactions with TOC in the cytosol (May and Soll 2000). 14-3-3 proteins are a family of regulatory proteins. They facilitate signal transduction and protein translocation, are regulated by the proteasome degradation pathway to modulate stress and growth, and may have a role in the protein quality control system (Gökirmak et al. 2010; Sato et al. 2011; Yuan et al. 2003). 14-3-3 proteins also have chaperone-like functions. Their holdase activity prevents protein aggregation and corrects misfolded proteins. A significant number of chloroplast protein transit peptides contain a 14-3-3 phosphopeptide binding motif. May and Soll found that certain phosphorylatable precursors in complex with 14-3-3 and cytosolic Hsp70 were more competent (3- to 4-fold) for import than uncomplexed precursors. Also, the complex may be useful in determining substrate specificity to the chloroplast or mitochondria. While the complex increases transport competency, mutations to the 14-3-3 transit peptide binding site did not disturb chloroplast import (Nakrieko et al. 2004). In addition to Hsp70 and 14-3-3, the Hsp90 chaperone assists precursors in the cytosol. Most precursors associate with Hsp70 (Ruprecht et al. 2010; Zhang and Glaser 2002), but a sub-class 4

of those precursors also associate with Hsp90 such as pNTT, pPC1, and pOE33 (Qbadou et al. 2006). Hsp90 chaperone activity is assisted by HOP (Hsp70/Hsp90 Organizing Protein) cochaperone and the immunophilin, FKBP73. Together they form a guidance complex with the precursor. Following translation, Hsp70 is predicted to bind precursors to prevent aggregation. HOP, as a part of the guidance complex, is predicted to transfer the precursors from Hsp70 to Hsp90. The complex is then predicted to interact with TOC 64. However, the formation of this complex is not essential to chloroplast import because in a Toc64 knockout study chloroplast import efficiency levels were not affected by the absence Toc64 (Hoffman and Theg 2005). In the stroma, Hsp70 and Hsp93 (from the Clp family), are ATPases that associate with substrates during the late stage of transport as the precursor emerges from the Tic translocon likely playing a role in pulling precursors across the envelope (Flores-Pérez and Jarvis 2013). Hsp70 is needed for effective transport across the chloroplast envelopes Immunoprecipitation studies showed that plastid Hsp70 precipitated Tic110, Hsp93 and the precursor, suggesting that it operates in a complex (Shi and Theg 2010). Hsp93 is predicted to be a motor for precursor transport mainly because it co-precipitates with TOC and TIC components (Akita et al. 1997; Nielsen et al. 1997; Chou et al., 2003; Rosano et al. 2011), binds the transit peptides of precursor substrates (Huang et al. 2015), and, when absent, is lethal during embryo development or can simply impair import (Constan et al., 2004; Kovacheva et al., 2007). Hsp93 has proteolytic activity by way of its Clp (caseinolytic protease) core. Attempts to distinguish the chaperone and proteolytic activity of Hsp93 during import showed that expression of Clp-inactive Hsp93 was not able to rescue chloroplast deficient in Hsp93. This demonstrated the importance of Clp in chloroplast import and suggests a quality control function for Hsp93, rather than a motor function (Flores-Pérez et al. 2016).

1.3 TOC

1.31 TOC Components Toc75 (75 kDa) is a membrane protein located in the outer envelope of chloroplasts. It is a component of TOC (Figure 1.2) and predicted to make up the translocating channel. Toc75 originates from cyanobacteria, but is nuclear-encoded and translated in the cytosol. Import into the chloroplast is carried out through the general import system (Hofmann and Theg 2005). Toc75 has a N-terminal bipartite signal containing a transit peptide and a glycine-rich segment (Tranel et al., 1995; Tranel and Keegstra 1996; Inoue et al. 2001; Inoue and Keegstra 2003). The transit peptide directs Toc75 to the stroma and the glycine-rich segment helps it integrate into the outer envelope membrane (Inoue and Keegstra 2003). Toc75 is also characterized by a C-terminal β- barrel domain (~45 kDa) and three hydrophilic polypeptide-transport-associated (POTRA) domains on the N-terminus (Sveshnikova et al. 2000; Arnold et al. 2010; Koenig et al. 2010; Paila et al. 2015). Toc75 is a member of the outer membrane protein of 85 kDa (Omp85) superfamily (Hsu and Inoue 2009). The Omp85 superfamily are bacterial outer membrane proteins (OMP) and are responsible for OMP assembly and protein translocation (Voulhoux and Tommassen 2004, Schleiff et al. 2011). Omp85 members are composed of a 16-stranded C-terminal β-barrel and up to five POTRA domains that have β1α1α2β2β3 secondary structures (Koenig et al. 2010; Paila et al. 2015). The role of the Toc75 POTRA domains are not fully defined. The function of other Omp85 superfamily proteins, BamA and Sam50, point to possible roles for Toc75 POTRA domains. BamA

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POTRA repeats can interact with an important periplasmic chaperone called SurA, other nascent OM proteins, and components of the BAM complex (BamB-E) (Bennion et al., 2010; Kim et al., 2007, Noinaj et al., 2015). BamA POTRA domains are essential for inserting β-barrels in the OM of E. coli. The POTRA domains may interact with the C-terminal domain of the β-barrels, acting as a gate for the membrane channel (Noinaj et al. 2013; Bakelar et al. 2016). The Sam50 POTRA domain also functions in protein-protein interactions to help facilitate import. In mitochondria, the Sam50 POTRA domain was found to interact with β-barrel precursors in the IMS to release them from the SAM complex and integrate them into the outer membrane (Kutik et al., 2008, Stroud et al., 2011). From this, it is likely that Toc75 POTRA domains have multiple roles interacting with proteins, to help assemble membrane biogenesis machinery (Koenig et al., 2010). A number of studies (Sommer 2011; Hsu 2012; Paila et al. 2016) have investigated the role of Toc75 POTRA domains by studying its topology. Two possibilities were explored for the topology of the N-terminal POTRA domains where they could be localized to the IMS or the cytosol. If the POTRA domains were localized to the cytosol, they could interact with chaperones and other cytosolic factors to assist precursor folding and transit peptide recognition. If they were localized to the IMS, they could bind precursors and hand them off to the TIC complex. Recently, Toc75 POTRA domains were found in the IMS from yellow fluorescent protein (YFP) assays and immunogold electron microscopy (Chen et al. 2016). Toc75 POTRA domains seem to have multiple roles related to TOC assembly, precursor import and chaperone recruitment (Paila et al. 2016). Deletions of one or more of the Toc75 POTRA repeats produced negative phenotypes in Arabidopsis thaliana (Paila et al. 2016). Specific deletions changed the distribution and size of the TOC complex on the outer membrane and disrupted precursor import into the chloroplast. Lastly, specific interactions were observed between POTRA repeats and two IMS chaperones, Tic-III and Tic-IV. Interactions were also observed between the POTRA repeats and precursors. The multiple functions of Toc75 POTRA domains are consistent with the activity of the other Omp85 family proteins. Toc34 is a 34 kDa transmembrane protein and one of two GTPases that work with the Toc75 channel to import precursors (Schleiff and Becker 2011; Lumme et al. 2014). It is composed of a single C-terminal transmembrane domain (TMD), a short C-terminal sequence (CTS) toward the TMD, and an N-terminal domain that’s exposed to the cytosol. The N-terminal domain includes a G-domain that hydrolyzes GTP. Toc34 binds the transit peptide of precursors and transfers them to Toc75, using its GTPase activity (Kessler and Schnell 2002; Lee et al. 2009). The function of Toc34 is accomplished using a GTP switch. GTP switches are unidirectional GTP hydrolysis mechanisms that regulate signal transduction. Bona fide GTPases are regulated by GTPase-activating proteins (GAPs) and Guanine Nucleotide Exchange Factors (GEFs). They regulate intermolecular structure transitions that determine whether the switch is on or off. GAPs move the switch from the active GTP-bound form to the inactive GDP-bound form. Once the switch is off, GEFs can turn it on again by inducing the release of GDP so the GTPase can associate with another GTP. Toc34 is classified in a subgroup of GTPases (GAD proteins) that are regulated by nucleotide- dependent dimerization instead of by GAPs and GEFs (Gasper 2009). Unlike standard GTPases which are structurally induced by GTP hydrolysis, the dimer dynamics of Toc34 is induced by substrate and nucleotide binding. Substrate-dependent dimer flexibility could be a general regulatory mode for GTPases that dimerize (Lumme et al. 2014). For example, Toc34 dimers alter between two main states in the GTP cycle, the tight GDP-loaded and the flexible GTP-loaded 6

state (Lumme et al. 2014). Biophysical experiments, FRET and PELDOR, determined the structural distances between the dimers at all phases of the GTP cycle and showed there is varying dimer flexibility throughout the cycle but no dimer dissociation was detected. The transition between the states are induced by substrate binding and are nucleotide-dependent. The binding of the transit peptide to the GDP-loaded Toc34, induces a slight opening of the dimer which is assumed to lead to the dissociation of GDP to allow for another GTP to come in the catalytic site. Nucleotide-dependent dimerization may allow Toc34 to bind the transit peptide of precursors and transfer them to Toc75 (Aronsson and Jarvis 2011; Richardson et al. 2014). Transit peptide binding regulates dimerization which is linked to GTPase activity. The GDP-loaded Toc34 dimer can recognize precursors (Sommer and Schleiff, 2009; Lumme et al. 2014) and induce an opening in the dimer (Lumme et al. 2014). Nucleotides could then be exchanged in the catalytic sites (Oreb et al., 2011; Lumme et al. 2014), further opening the dimer (Lumme et al. 2014) and resulting in a GTP-bound state. In this open conformation, it has been speculated that Toc34 transfers the precursor to the second GTPase, Toc159. Bauer et al., 2002; Becker et al., 2004 found that Toc34 forms a homodimer with itself and a heterodimer with Toc159 (Bauer et al., 2002; Becker et al., 2004). Becker 2004 suggested that monomeric Toc34 has a higher affinity for Toc159 in the presence of precursor. Toc159 is the second GTPase in the TOC system and works with its homologue, Toc34, to recognize precursors. Toc159’s ability to transition between homodimers and monomers is thought to be essential for precursor recognition (Chang et al. 2017). In addition, it may act as the initial receptor for precursors (Perry and Keegstra 1994; Ma 1996; Chang et al. 2017) and is essential for chloroplast biogenesis in A. thaliana (Bauer et al. 2002). Mutants lacking Toc159 were albino, could not survive on soil, and their photosynthetic proteins were transcriptionally repressed (Bauer et al. 2000). Toc159 is composed of a C-terminal transmembrane domain (M) anchored in the outer envelope membrane, a GTPase domain (G) and an acidic N-terminal domain (A) in the cytosol (Hirsch et al. 1994; Chen et al. 2000). Like Toc34, Toc159 uses its G domain to directly bind precursors (Smith et al. 2004). The G-domain binds chloroplasts in vitro and may have intrinsic targeting information (Smith et al. 2002). The M-domain also contains targeting information and a truncated M-domain can target itself to and integrate into the outer envelope (Lee et al. 2003). A receptor role dealing with precursor specificity has been suggested for the A-domain because it is unstructured and divergent among Toc159 isoforms (Inoue et al. 2010). A role mediating TOC assembly has also been suggested because of the A-domain’s structure (Richardson et al. 2009). Toc159 is different from other outer envelope proteins because of its novel sorting signal which varies from most outer membrane proteins (OMP) and shares properties with chloroplast transit peptides (Lung and Chuong 2012; Lung et al. 2014). Bioinformatic analysis showed physicochemical and structural similarity to chloroplast transit peptides (Lung and Chuong 2012). Using fluorescent protein-tagging, the Toc159 sorting signal was shown to, not only, target fusion proteins to the chloroplast, but undergo cleavage after translocation to the stroma like chloroplast transit peptides (Lung and Chuong 2012). In addition to sharing properties with chloroplast transit peptides, the Toc159 sorting signal varies from most OMPs, which carry their sorting information in the transmembrane domain (Hofmann and Theg 2005; Bölter and Soll 2011; Lee et al. 2013) or a single hydrophobic alpha helix (alpha- helical OMPs) (Hofmann and Theg 2005; Bölter and Soll 2011). The Toc159 mostly resembles a 7

subset of alpha helix OMPS, tail-anchored proteins, that carry the sorting signal at the C-terminus (Fischer et al. 1994; Chen and Schnell 1997; Froehlich et al. 2001; Dhanoa et al. 2010; Kutay et al. 1993; Abell and Mullen 2011). In Bienertia sinuspersici, Toc159 has OM sorting information in its C-terminal (56-residue) sorting signal, however lacks the hydrophobic alpha-helix and OM insertion signal seen in tail-anchored proteins. Unlike tail-anchored proteins, the Toc159 sorting signal can mediate targeting to the chloroplast surface (Lung and Chuong 2012; Lung et al. 2014). While the sorting signal for Toc159 is novel, the function of Toc159 seems to be regulated similar to Toc34. Toc159 has also been shown to use dimerization (Yeh et al. 2007) and to bind the transit peptide of precursors with its G-domain (Smith et al. 2004). To investigate the mechanism on a molecular level, Chang et al (2017) looked at tertiary and quaternary Toc159 structures that directly interact with precursors. They were able to map precursor binding sites on the Toc159 homodimer using cysteine-cysteine cross-linking and protease treatments. They found the site was located on the switch II region of the GTPase domain and on residues at the dimer interface (Chang et al. 2017). The transit peptide was found in close proximity to both regions whereas the mature domain of the precursor was only associated with residues at the dimer interface (Chang et al. 2017). They also showed that the dimer dissociates before contacting the precursor at the interface region (Chang et al. 2017). Similar to Toc34, dimerization and precursor recognition are coupled processes.

1.32 The TOC Model The ~800 kDa TOC complex (Figure 1.2) exists in an approximate 4:4:1 or 3:3:1 (Toc75:Toc34:Toc159) stoichiometric ratio (Schleiff et al., 2003; Kikuchi et al., 2006; Chen and Li, 2007). At the start of import, precursor proteins interact with the two GTPase receptors, Toc34 and Toc159. Transit peptide binding to the G-domains alters Toc34 and Toc159 dimer conformation, leading to nucleotide exchange and GTP hydrolysis. GTP hydrolysis allows the precursor to be transferred to the β-barrel channel, Toc75, and translocated to the IMS. Toc75 POTRA domains mediate precursor transfer to TIC by recruiting two IMS chaperones, Tic-III and Tic-IV (Aronsson and Jarvis 2011; Shi and Theg 2013; Richardson et al. 2014).

1.4 TIC

1.41 TIC Components In contrast to TOC, less is unknown about the protein constituents and the mechanism of the TIC translocon (Figure 1.2). A number of TIC candidates (Tic20, Tic21, Tic40, Tic56, Tic100, Tic110 and Tic214) have been identified and some of these proteins have been proposed to have a role in import. The classical model of TIC includes Tic22, Tic110, Tic40, and Tic20 as possible translocon components and the newest model includes Tic20, Tic56, Tic100 and Tic214 as a part of a recently identified 1-mega Dalton complex. Tic22 is an IMS protein bound to the inner envelope membrane and an essential component of the TIC complex (Kouranov et al. 1998; Rudolf et al. 2013). A study by Rudolf et al. demonstrated Tic22’s essential role by evaluating the function of its isoforms, Tic22-III and Tic22-IV, in Arabidopsis thaliana. T-DNA insertion lines were generated for both isoforms. Individual mutations had minimal on phenotype, more than likely due to Tic22 redundancy. Double T-DNA insertion mutations, however, decreased growth, import rate and photosynthetic performance in Arabidopsis thaliana. Further delineating its role in the TIC complex, Tic22 was proposed to mediate the transfer of precursors from TOC to TIC (Kouranov et al. 1998). Tic22’s role as a 8

mediator or scaffold was supported in crosslinking studies between it and translocating precursors (Ma et al. 1996; Kouranov and Schnell 1997; Kouranov et al. 1998). Crosslinks between precursors and Tic22 were detected during the late stage of import, when precursors are in the process of being pulled through the inner envelope membrane (Ma et al. 1996; Kouranov and Schnell 1997). Tic110, an abundant inner envelope protein, was suspected to be directly involved in TIC translocation because of its association with translocon intermediates (Kessler and Blobel 1996; Nakai 2015). Tic110 has a large C-terminal domain situated in the stroma and two N-terminal helices segments that anchor it in the inner envelope (Jackson et al. 1998; Inaba et al. 2005; Balsera et al. 2009; Tsai et al. 2013; Nakai 2015). On the C-terminal half, it has several elongated HEAT-repeats, similar to HEAT-repeat motifs that act as scaffolds in protein-protein interactions (Nakai 2015). A scaffolding function was proposed for Tic110 in stabilizing emerging precursors at the N-terminus and recruiting stromal chaperones, Hsp93 and cpHsp70, to help pull the precursor across the IM (Kessler and Blobel 1996; Jackson et al. 1998; Inaba et al. 2003). Tic40 is an integral membrane protein in the inner envelope membrane and a component of the TIC complex (Stahl et al. 1999). Tic40 is embedded in the inner membrane envelope by a single N- terminal TMD (Stahl et al. 1999) and has a large soluble domain that extends into the stroma (Chou et al. 2003). A function as a co-chaperone was proposed for Tic40 due to structures on its C-terminal stromal domain (Chou et al. 2003). There are two binding sites on the stromal domain, a tetratricopeptide (TPR) repeat followed by a Hip/Hop/Sti1 domain. Because of their similarity to co-chaperones St1p/Hop and Hip, they are proposed to be a binding site for Hsp70 and Hsp90 (Höfeld et al. 1995; Frydman and Höhfeld 1997; Scheufler et al. 2000; Chou et al. 2003). In addition to the analysis of the binding sites, Chou et al. were able to co-immunoprecipitate Tic110 and Hsp93 along with Tic40. Together, this suggests a function for Tic40, providing a scaffold for stromal chaperones along with Tic110. Tic21 is a component of the TIC complex and is essential to import across the inner envelope membrane (Teng et al. 2006). Tic21 is a 21 kDa integral membrane protein that is embedded in the inner envelope and is predicted to have four transmembrane helices. The function of Tic21 is unclear but it’s structure is similar to Tic20. Knockout mutants for Tic21 and Tic20 also share the same phenotype, suggesting they perform a similar function in biogenesis (Teng et al. 2006). Tic21 is also weakly associated with the 1MDa complex containing Tic20 (Kikuchi et al. 2015), suggesting a role facilitating import. Tic56, another TIC component, is a 56 kDa nuclear-encoded protein with no predicted transmembrane helices. Tic56 is also thought to be embedded in the 1 MDa complex (Kikuchi et al. 2013). Tic56 is an important component of TIC and a part of the 1 MDa complex, but it is not essential to import across the inner envelope membrane. In the absence of Tic56, plants have diminished growth and display an albino phenotype but are still able to import proteins (Köhler et al. 2015). Because the absence of Tic56 causes a decrease in the 1 MDa complex (Kikuchi et al. 2013), it is likely that other translocons are available in the inner envelope membrane to import precursors (Köhler et al. 2015). Tic100 is another TIC component. It is a 100 kDa nuclear-encoded protein and resides on the periphery of the 1 MDa complex, facing towards the IMS (Kikuchi et al. 2013). It has three short Membrane Occupation and Recognition Nexus (MORN) motifs that may help facilitate membrane interactions.

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Tic214 is a chloroplast- encoded protein (Ycf1) with six predicted transmembrane domains in the N-terminus (Kikuchi et al. 2013). While Tic214 is essential to the assembly and function of the 1 MDa complex, it is not essential for plastid protein import and accumulation (Bölter and Soll 2017). All monocotyledonous plants and some dicotyledonous plants lack Tic214 (de Vries et al. 2015). One study using spectinomycin treatments demonstrated that when translation is inhibited, including Tic214 synthesis, in plastids, protein translocation still occurs. While Tic214 is important for plant growth in Chlamydomonas and Figure 1.2 A MODEL OF TOC AND TIC. Tobacco (Boudreau et al. Most nuclear-encoded proteins are imported into the chloroplast by TOC (Translocon 1997; Drescher et al. 2000), at the Outer Chloroplast Envelope Membrane) and TIC (Translocon at the Inner and the function of the 1 Chloroplast Envelope Membrane). TOC and TIC work in tandem to transport MDa complex, it is not a preproteins across the two envelopes. Preproteins are recognized by the two GTPase receptors, Toc34 and Toc159. Both receptors recognize the transit peptide (orange) general import component. with their G-domain and undergo conformational changes to exchange nucleotides. Tic20 is also an integral (Toc159 is equipped with an A-domain that may help with preprotein specificity.) membrane protein in the Nucleotide hydrolysis helps transfer the preprotein to the Toc75 channel and. in a inner envelope membrane partially unfolded state, it is transported into the IMS (inner membrane space). Tic22 and a component of the TIC mediates the transfer of the preprotein to the TIC complex in the IMS. In the inner envelope, the preprotein interacts with the 1-megadalton complex. The complex is complex (van Dooren et al. composed of the potential channel, Tic20, and three membrane spanning proteins, 2008; Kouranov et al. 1998). Tic214, Tic100, and Tic56. Tic21, Tic40, and Tic110 were found in association with the Tic20 is predicted to span 1-megadalton complex. A scaffolding and energetic role is proposed for Tic40 and the membrane four times, Tic110 due to their structures (TPR & Hip/Hop/Sti1; Helix repeats) and associations has both of its tail ends with Hsp70 and Hsp93. No clear role has been identified for Tic21, however it is essential for preprotein import and a main component of TIC (adapted from Jarvis and exposed to the stroma, and Robinson 2004). is composed of mostly alpha-helices (Machettira et al. 2011; van Dooren et al. 2008; Kovács-Bogdán et al. 2011). In past studies, Tic20 has formed crosslinks with importing precursors and associated with TOC/TIC components (Ma et al. 1996; Kouranov et al. 1998). In addition to being a part of the TIC complex, Tic20 may form a protein-conducting channel for the TIC complex. Kovács-Bogdán et al. showed that Tic20 can form a cation-selective channel and Kikuchi et al. identified Tic20 as a core component in a 1-megadalton translocation complex. 10

1.42 The TIC Model A TIC model (the classical model) was proposed (Kessler and Schnell 2009; Schwenkert et al. 2011; Li and Chiu 2010; Shi and Theg 2013; Nakai 2015) involving the IMS protein Tic22, an association of TIC membrane proteins that formed the translocation channel, and a motor complex consisting of two heat shock proteins and two TIC membrane proteins. In the beginning of the model, Tic22 interacts with the N-terminus of precursors, acting as a scaffold between TOC and TIC. It is responsible for transferring precursors to Tic110. The translocation channel consists of transient associations between Tic110, Tic20, and Tic21. During the late stage of import, the N-terminal of the precursor interacts with co-chaperones in the stroma that pull it across the membrane. The N-terminal interacts with soluble domain of Tic110, and together, along with Tic40, Hsp93 and cpHsp70 are recruited. The N-terminal is transferred to the chaperone proteins which pulls the precursor into the stroma using ATP hydrolysis. This model is not supported by strong evidence. Tight association between the TIC candidates (Tic110, Tic20, and Tic21) and precursors was not found. Also, evidence of a transient TIC complex was not found. The strongest evidence for a TIC translocon complex is a 1MDa complex (Figure 1.2), composed of Tic56, Tic100, Tic214, and the core component Tic20 (Kikuchi et al. 2013). Direct binding with precursors was demonstrated in a pull-down assay incorporating the IgG-binding domain of Protein A (Kikuchi et al. 2013). Tic214 and Tic20 could have roles in forming the structure of the channel. Tic20 is suspected because it makes up the core of the complex and is capable of forming cation-selective channels. Tic214 may be a part of the channel because of its N-terminal transmembrane helices which are predicted to span the membrane six times (Nakai 2015). Tic100 is on the periphery of the 1 MDa complex, facing the IMS, and may interact with the TOC complex or act as precursor receptors. Like the Classical Model, Tic110 and Tic40 may work together to recruit heat shock proteins to pull precursors into the stroma. However, because the absence of Tic110 and Tic40 did not disrupt import across the inner envelope membrane, other co- chaperones and heat shock proteins need to be evaluated to delineate the motor for the TIC complex.

1.5 SRP The Signal Recognition Particle (SRP) pathway is a highly conserved pathway that co- translationally targets polypeptides for secretion. While its size and composition differ between organisms, the SRP pathway has two conserved components: the SRP54 (54 kDa) protein and the SRP RNA. The SRP receptor, which is anchored in the membrane, also has a key function in the pathway. The SRP pathway is powered by and protein targeting is regulated by GTP hydrolysis. GTP hydrolysis is triggered by the interaction of GTP-binding domains on both SRP54 and the SRP receptor. The SRP pathway exists in bacteria and plastids, however in plastid chloroplasts, the SRP RNA is absent although highly conserved elsewhere. In bacteria, SRP54/Ffh and SRP RNA co-translationally target proteins to the plasma membrane. Ffh is comprised of three domains. There is a four-helix bundle at the N-terminal (N) domain, followed by a GTP-binding (G) domain, and a methionine rich C-terminal (M) domain (Zopf et al. 1990; Freymann et al. 1997; Ziehe et al. 2017). Together the N and G domains form the NG domain, the functional unit of GTP hydrolysis. The second component of the SRP pathway, SRP RNA, is a conserved 4.5S RNA ribonucleotide (Ziehe et al. 2017). SRP RNA is elongated and contains two internal loops and one conserved tetraloop. Together, SRP RNA and SRP54/Ffh form the SRP complex. Within the complex, the internal loops of SRP RNA result in high affinity

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binding between it and the M domain of SRP54/Ffh. In co-translational targeting, the M domain of SRP54/Ffh recognizes the nascent polypeptide chain emerging from the ribosome. The SRP complex guides the entire ribosome nascent chain complex (RNC) to the SRP receptor (FtsY) on the plasma membrane. Like SRP54/Ffh, the FtsY receptor has an NG domain (Freymann et al. 1997; Montoya et al. 1997; Ziehe et al. 2017). A complex is formed between FtsY and the RNC- bound SRP complex through interactions between both of their NG domains (Egea et al. 2004; Focia et al. 2004). The formation of the complex is accelerated by interactions between the tetraloop of SRP RNA and the Fts Y receptor. FtsY/SRP complex formation is accelerated by ~100- to ~400-fold (Ziehe et al. 2017). The FtsY receptor only interacts with the RNC-containing SRP when both it and SRP54/Ffh G domains are bound with GTP. Both Ffh and FtsY NG domains undergo conformational changes to induce GTPase activity (Jagath et al. 2000; Shan and Walter 2003). GTP hydrolysis powers the dissociation of the SRP/FtsY complex and the release of the RNC to the secretory pathway (Akopian et al. 2013; Denks et al. 2014). The RNC is handed off to the SecYEG/YidC translocon where it can be integrated into or translocated across the plasma membrane. In spermatophytes and higher plants, the SRP pathway (cpSRP) operates on the thylakoid membrane of chloroplasts. Like the bacterial pathway, it consists of a SRP54/Ffh homolog (cpSRP54), a homolog for the receptor (cpFtsY), which is embedded in the thylakoid membrane. The cpSRP pathway also transfers its substrates to the chloroplast secretory system (cpSecY1, cpSecE1, and Alb3) (Zhang et al., 2001; Walter et al., 2015b). However, unlike in bacteria, the cpSRP pathway lacks a conserved SRP RNA. The SRP RNA is an important component of the SRP complex in bacteria, binding the Ffh protein, recognizing the RNC, interacting with the receptor FtsY and accelerating the formation of the FtsY/SRP complex. Despite the absence of the SRP RNA, the kinetics of the cpSRP pathway are ~400-fold faster (Jaru-Ampornpan et al. 2007). The cpSRP pathway is compensated by a “pre-organized” cpFtsY NG domain, which produces a 5- to 10-fold more efficient interaction between the cpFtsY and cpSRP54 (Jaru- Ampornpan et al., 2007; Stengel et al., 2007; Chandrasekar et al., 2008). The domain is in a closed conformation and minimizes conformational rearrangements during the formation of the cpSRP/cpFtsY complex (Stengel et al., 2007; Chandrasekar et al., 2008). The M domain of cpSRP also helps compensates the absence of SRP RNA. It accelerates and stabilizes the cpSRP54/FtsY complex formation by ~100- to ~200-fold (Chandrasekar et al., 2017). The M domain interacts directly with the receptor through a binding interface. The binding interface is characterized by basic residues in the cpFtsY G domain and one negatively charged region at the N-terminus of the cpSRP54 M domain (Chandrasekar et al., 2017). The evolutionary appearance of the binding interface occurred alongside the loss of the tetraloop SRP RNA components within the green lineage (Chandrasekar et al., 2017). The cpSRP54 targets substrates in the stroma through (1) an association with the 70S ribosome and (2) by forming a complex with cpSRP43 (Franklin and Hoffman, 1993; Schünemann et al., 1998; Klimyuk et al., 1999; Groves et al., 2001; Hermkes et al., 2006; Holdermann et al., 2012). In its association with the 70S ribosome, cpSRP54 has some significance but no central role in co-translational integration. Transient interaction was observed in a crosslinking experiment between the ribosome-associated cpSRP54 and the nascent chain of photosystem II reaction center protein D1 (Nilsson et al., 1999; Nilsson and van Wijk, 2002). This demonstrated that the complex can integrate plastid-encoded proteins. However, a deletion mutant of cpSRP54 produced mild effects of decreased levels of plastid-encoded thylakoid membrane proteins (Amin et al., 1999). So far, the studies do not point to a central role for cpSRP54 in protein co- 12

translational integration. In contrast, cpFtsY is essential to protein integration. Deletion mutants of cpFtsY produced phenotypes such as seedling lethality, impaired photosystem II repair cycles, and chlorosis (Tzvetkova-Chevolleau et al., 2007; Asakura et al., 2008; Walter et al., 2015a). The cpFtsY receptor was shown to be a component of the D1 protein RNC complex and has direct involvement in the insertion of protein D1 (Walter et al., 2015b). The second way the cpSRP pathway targets posttranslational substrates is by forming a heterodimer (the cpSRP complex) with the stromal protein, cpSRP43 (Aldridge et al., 2009; Grudnik et al., 2009; Richter et al., 2010; Akopian et al., 2013b). This mode of targeting is specifically for nuclear-encoded light harvesting chlorophyll a/b (LHCP) binding proteins. The tail region of cpSRP54 contains a conserved cpSRP43 binding motif which interacts with the cpSRP43 chromodomain 2 to form the heterodimer (Funke et al., 2005; Holdermann et al., 2012). The resulting cpSRP complex forms the transit complex by binding the LHCP substrate. In addition to being a part of the cpSRP complex, cpSRP43 acts as an LHCP chaperone by preventing aggregation (Falk and Sinning, 2010; Jaru-Ampornpan et al., 2010). The interaction between the ankyrin repeat region of cpSRP43 and a conserved 18 amino acid L18 motif between the second and third TMD of the LHCP are responsible for the formation of the transit complex (DeLille et al., 2000; Tu et al., 2000; Jonas-Straube et al., 2001; Stengel et al., 2008). cpSRP54 may also help form the transit complex using its M domain to directly interact with the third TMD region of LHCP (Henderson et al., 2016). The cpFtsY receptor also has an essential function, along with cpSRP54, in driving protein integration. When the transit complex delivers the LHCP to cpFtsY and Alb3, GTPase activity from cpSRP54 and cpFtsY drive the integration of the LHCP (Richter et al., 2010). In the cpSRP pathway, LHCP insertion is optimal with cpSRP54, cpSRP43, and cpFtsY. However, cpSRP43 was shown to transport LHCPs without the presence of cpSRP54 or cpFtsY, at a slightly reduced efficiency (Tzvetkova- Chevolleau et al., 2007). Although insertion can occur without it, cpSRP54 was shown to structurally enhance the affinity between cpSRP43 and LHCPs (Gao et al., 2015; Liang et al., 2016).

1.6 SECRETORY SYSTEM The Secretory (Sec) pathway is responsible for transporting and integrating proteins in both bacteria and plastids. Harnessing the energy from nucleotide hydrolysis, the Sec pathway transports unfolded proteins. The Sec pathway can be found in the eukaryotic endoplasmic reticulum (ER), the plasma membrane of archaea and eubacteria, and the thylakoid membranes of plants and algae (Osborne et al. 2005; Bolhuis 2004; Veenendaal et al. 2004; Dalbey and Chen 2004; Vrontou and Economou 2004). The Sec pathway consists of a protein conducting channel and a number of accessory factors, including chaperones and components of the SRP pathway. For bacterial integration, some substrates are targeted co-translationally using SRP54, Ffh, and the FtsY receptor. The SRP components are able to guide the RNC to the Sec translocon (Akopian et al. 2013; Denks et al. 2014). In bacteria, the translocon (SecYEG) is composed of SecY, SecE, and SecG. SecY and SecG are multi-spanning membrane proteins while SecE is a single-spanning membrane protein. An ATPase, SecA, is also a component of the Sec pathway where it drives translocation across the membrane. SecA binds the SecYEG channel as well as the signal peptide and mature domain of the precursor. It undergoes conformational changes linked to ATP hydrolysis to thread ~20 to ~30 peptide segments of the precursor through the SecYEG. The chloroplast Sec (cpSec) 13

pathway shares similarities with the E. coli Sec system because of chloroplasts evolutionary link to bacteria. The cpSec channel is composed of cpSecY and cpSecE, however it differs from the bacterial pathway because it lacks a homologue for SecG (Cline 2003; Mori and Cline 2001). And similar to bacterial systems, transport is powered by cpSecA. Crosslinking studies also showed associations between the precursor, cpSecA, and parts of the translocon. Other accessory factors in the cpSec pathway are from the cpSRP system (cpSRP, cpFtsY, YidC and cpSRP43), which help target substrates to the cpSecYEG translocon (Gerdes et al. 2006; Asakura et al. 2004). The Sec pathway is also responsible for integrating membrane proteins into the bilayer. In E. coli, membrane proteins are integrated co-translationally during elongation on the ribosome (Huber et al. 2011). SRP components target the nascent chain and ribosome to the SecYEG translocon (Ulbrandt et al., 1997). Integration into the bilayer is driven by the SecA ATPase and the GTPase on the ribosome (Asakura et al. 2004). YidC also works with the Sec components, as an insertase, to integrate membrane proteins into the bilayer. In chloroplasts and plastids, membrane proteins can also be integrated through the cpSec pathway. Many membrane proteins are multi-spanning, ligated to cofactors, and assembled within the core photosystems. These characteristics have made it challenging to study them in isolated thylakoids because of difficulties with their assembly. Because of this, indirect evidence has been used to delineate the cpSec integration pathway. A pathway containing cpSecA, cpSRP components, the ribosome, and cpSecYE, similar to bacteria, has been suggested for the cpSec integration pathway (Cline and Theg 2007). Two membrane proteins, Cytf and PsbA, were used to investigate the components of the cpSec integration pathway. PsbA is a multi-spanning protein and Cytf is a single-spanning protein with a large luminal domain. Evidence for the involvement of cpSecA was found with In vitro reconstitution (Mould et al. 2001; Röhl and Van Wijk 2001; Cline and Theg 2007) and cpSecA- null mutant (Voelker and Barkan 1995) assays with Cytf in maize. With the cpSecA-null mutants, an accumulation of Cytf precursors suggested that cpSecA may have a role in driving integration. Ambiguous data on SRP components with Cytf and PsbA was acquired that may imply its role targeting proteins to the pathway. Crosslinking between cpSRP54 and PsbA was observed in an organelle-free translation system (Nilsson et al. 1999). cpSRP54 also formed crosslinks to the signal peptide of Cytf in a wheatgerm translation system (High et al. 1997), however no crosslinks were observed using a chloroplast translation system in a different study (Röhl and Van Wijk 2001). The use of the cpSecYE channel was implied for Cytf and PsbA. Since booth proteins are translated on bound thylakoid ribosomes, they are integrated co-translationally. The nascent chains of PsbA were found to be associated with cpSecY and ribosomes, suggesting its translation is coupled to the cpSec pathway (Zhang et al. 2001). Lastly, in Chlamydomonas reinhardtii, the presence of the accessory factor Alb3 is needed for the efficient assembly of D1 into PSII reaction centers (Ossenbühl et al. 2004). Together, cpSecYE, the ribosome, cpSRP54 and Alb3 are implied in the configuration of the cpSec pathway for integrating membrane proteins.

1.7 TWIN ARGININE TRANSLOCATION SYSTEM The twin arginine translocation (Tat) pathway is prevalent in prokaryotes and is therefore an ancestral translocase (Berks 1996; Dilks et al. 2003). It is found in the chloroplasts of plants and algae, the mitochondria of some archaea and most bacteria (Cline and Theg 2007). The Tat system is involved in energy production, respiratory and photosynthetic, symbiosis and pathogenesis in plants and animals.

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The Tat system is distinctive in its ability to transport fully folded proteins, of various sizes, using the proton motive force (Berks et al. 2000; Rodrigue et al. 1999; DeLisa et al. 2003). Unlike Sec and TIC/TOC, the Tat system relies on the electrochemical gradient to power transport instead of nucleotide hydrolysis (Cline and Theg 2007). It is suggested that the Tat system may use a proton well to couple electron transport to transport, similar to the way ATP synthesis is coupled to electron transport in the electron transport chain (Mitchell 1968; Cline and Theg 2007). The Tat system, depending on the organism, can have from 1 to ~150 different substrates and all of them are transported in a folded conformation. The protein substrates range in size, 2 kDa-100 kDa, and length, ~20Å to ~70Å (Henry et al. 1994; Berks et al. 2003). Tat can transport heterodimers, cross-linked tetramers and engineered unstructured polypeptides (Rodrigue et al. 1999; Ma and Cline 2010; Cline and McCaffery 2007; Richter et al. 2007). The Tat system is able to transport a wide range of folded substrates without damaging the integrity of the membrane and dissipating the proton motive force (PMF). The reason the substrates are transported in folded conformations may be because some rapidly fold upon synthesis or import, a lack of folding chaperones on the other side of the membrane or for a better control of cofactor specificity. The Tat system operates in a PMF-dependent cyclical process, using three membrane proteins, that create a temporary pore in the membrane to transport its folded substrates (Mori et al. 1999; Mori et al. 2001; Fincher et al. 2003). The three membrane proteins operate in a dynamic fashion in response to the PMF and the presence of the signal peptide on the protein substrates (Figure 1.3). The Tat system name was coined referencing the required twin arginine (RR) motif on the N-terminus of the signal peptide that is recognized by some of the membrane proteins. The signal peptide has signal peptidase recognition site towards the polar carboxyl-terminus, a central hydrophobic core (h-region) and a positively charged N-terminus which holds the twin arginine motif which is within the highly conserved consensus motif S/T-R-Rx-F-L-K (Cline and Henry 1996; Robinson and Bolhuis 2004). TatA, TatB, and TatC are integral membrane proteins that make up the components of the Tat system in bacteria (Settles et al. 1997; Sargent et al. 1998; Bogsch et al. 1998). In plants, the homologues are respectively named Tha4, Hcf106, and cpTatC. These proteins operate as multimers, in a cyclical and PMF-dependent fashion, to transport Tat substrates (Figure 1.3). TatA and TatB are highly homologous, having one short N-terminal transmembrane domain, a hinge region that intersects the top of the bilayer and the inner core, the amphipathic helix (APH) region that rests on top of the membrane and an unstructured C-terminus tail (Settles et al. 1997; Hu et al. 2010). The C-tail is longer for TatB. Both membrane proteins have an “L” shape due to the perpendicular orientation of the TMD and the APH. In contrast, TatC has a “glove” shape due to the folding pattern of its six transmembrane domains (TMD) (Ramasamy et al. 2013). The TMDs may be stabilized by two of its trans loops (1 and 2) which form an overhanging cap structure. Both its N- and C-terminus are oriented on the cis side of the membrane.

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The mechanism for transport beings with a receptor complex containing multimers of TatB and TatC in a 1:1 ratio (Kneuper et al., 2012; Tarry et al., 2009; Lee et al., 2006; McDevitt et al., 2005; Alami et al., 2003). The receptor complex is responsible for recognizing the twin arginine motif in the signal peptide of the precursor protein (Holzapfel et al. 2007; Gérard and Cline 2006). TatC is thought to be the core component and the scaffold due to it specifically binding the twin arginine motif and because Tat A and Tat B bind to it first before interacting with the precursor (Ramasamy et al. 2013). TatA exists in a separate pool of small oligomers. When the substrate binds the receptor complex and in the presence of the PMF, TatA oligomerizes within the cavity, or glove, of TatC (Dabney-Smith et al., 2006; Leake et al., 2008; Dabney-Smith and Cline, 2009; Kneuper et al. 2012). The oligomerized TatA, the receptor complex and the bound precursor together form the translocation complex. TatA is thought to be responsible for forming the pore due to it forming large oligomers, its abundance over TatB and TatC, its direct interaction with the precursor mature domain and it being required for the transport (Kostecki et al. 2010; Greene et al. Figure 1.3 A MECHANISTIC MODEL OF THE CHLOROPLAST 2007; Leake et al. 2008; Mori et al., TWIN ARGININE TRANSLOCATION (CPTAT) PATHWAY. 2001; Celedon and Cline, 2012; Stroma (S). Thylakoid Membrane (TM). Lumen (L). A. In a 1:1 ratio, Pal et al. 2012; Cline and Mori, cpTatC (green) and Hcf106 (pink) form the receptor complex which is 2001). After transport, the responsible for recognizing the transit peptide of the precursor. Tha4 translocation complex (dark pink) exists in small oligomers in the thylakoid membrane (T) and is predicted to form the pore during transport. B. In the presence of the disassembles back to the receptor proton motive force (PMF) the precursor binds the receptor complex and complex and the small pool of TatA Tha4 oligomerizes within the cavity of cpTatC. C. The precursor is (Mori et al., 1999; Cline and Mori, transported through the pore, and D. the mature protein is released in the 2001; Mori and Cline, 2002). lumen after the transit peptide is cleaved. E. The translocation complex disassembles back into the receptor complex and small oligomers of TatA is predicted to either form an Tha4. aqueous pore in the membrane or to weaken the integrity of the membrane enough to transport a substrate (Cline 2015; Gohlke et al. 2005; Cline and Theg 2007; Brüser and Sanders 2003). A charged zipper mechanism was explored, where the APH is inserted in the bilayer and held in

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place by salt bridges (Walther et al. 2013). Oligomers of TatA with inserted APH’s would support the aqueous pore model. However, APH insertion was not observed in two TatA topology studies (Koch et al. 2012; Aldridge et al. 2012). The latter mechanism, where the integrity of the membrane is destabilized, is better supported. A simulation of E. coli TatA in lipids showed oligomers of various sizes destabilizing the bilayer (Rodriguez et al. 2013). An NMR structural analysis of E. coli TatA showed that the TMD is responsible for weakening the membrane. Upon oligomerization, TatA undergoes a conformational change where the TMD is pulled towards the cytoplasm. As a result, the membrane is compressed and sensitized for rupture (Rodriguez et al. 2013). Another study showed the N-terminus and APH region of TatA also weakening the membrane in vivo (Hou et al. 2018). This evidence supports the transport model where TatA oligomerization causes hydrophobic mismatch and weakens the bilayer enough for substrate transport.

1.8 THESIS SUMMARY In this study, the composition of the cpTat translocon pore was further investigated by exploring direct binding between Hcf106 and the Oxygen Evolving Complex precursor (pOE17). Cysteine crosslinking was used to detect interactions between variants of pOE17 (pOE17-D68C, -S84C, - K99C, and -T115C) and Hcf106 (Hcf106-G6C, -G8C, -P10C, -L13C, -L14C, -V31C, -V38C, - R33C, -N34C, -G36C, -T38C, -L39C, -E48C, -S53C, -S58C, and -I64C. Direct binding was shown for three pOE17 variants (-D68C, -S84C, and -K99C) that had also shown various levels of interactions with Tha4 (Pal, et al., 2012) in the N-terminus, C-terminal APH region, and the C- terminus of Hcf106 (Pal, 2014). However, this study showed little to no direct binding between pOE17-T115C and the Hcf106 variants. In total, pOE17 interacts with Hcf106 at the N-terminus and even more so at the C-terminal APH region and C-terminus. Furthermore, whether cpTat substrates stay in contact with Hcf106 during transport was investigated. Crosslinking assays, followed by protease treatments, between Hcf106 and pOE17 were performed to determine whether the substrate was exposed to the membrane during transport. Unexpectedly, membrane- protected and -unprotected crosslinked products were indistinguishable from each other in the assay. The paper presents limitations with the study and directions for future research.

1.9 REFERENCES

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Chapter 2. INVESTIGATING THE PORE COMPOSITION OF THE CHLOROPLAST TWIN ARGININE TRANSPORT SYSTEM

Nefertiti Muhammad1, and Carole Dabney-Smith1 Department of Chemistry and Biochemistry and Cell, Molecular, and Structural Biology Graduate Program, Miami University, Oxford Ohio To be submitted to Biochemistry

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2.1 ABSTRACT We investigate the pore composition of the chloroplast Twin Arginine Transport system (cpTat). The cpTat system transports fully folded proteins into the thylakoid using the proton motive force and, together, cpTatC, Tha4, and Hcf106 transport precursors across the bilayer membrane. Tha4 is predicted to form the translocation pore; however, Hcf106 may also have a role because of the large size of some cpTat substrates and its homology to Tha4. In this study, direct binding between Hcf106 and a cpTat substrate, the Oxygen Evolving Complex precursor (pOE17), was investigated. Prior work showed direct binding to Tha4 for four variants of the substrate (pOE17- D68C, -S84C, -K99C, and -T115C) using cysteine crosslinking. The current study showed little to no direct binding between Hcf106 and the last variant of the substrate (pOE17-T115C); although, that variant could interact with Tha4. Together with precursor-Tha4 interaction maps, we concluded that both cpTat components bind directly to the substrate and the strongest interactions occur at the C-tail and the C-terminal end of the amphipathic helix (APH) region. This study also investigates whether cpTat substrates stay in contact with Hcf106 throughout transport. Protease treatments were performed on crosslinked Hcf106 and pOE17 at the C-tail and N-terminal residues of Hcf106. Unexpectedly, membrane-protected and -unprotected crosslinked products were detected in the N-terminus of Hcf106. The paper presents limitations with the study and directions for future research.

2.2 INTRODUCTION Chloroplast evolved from cyanobacteria which lived within eukaryote hosts as endosymbionts (Yoon et al. 2004). Over time, chloroplasts surrendered the majority of its genome to the host nucleus as a part of evolutionary gene transfer (Yoon et al. 2004). However, chloroplasts retained a portion of their original genome, which was stored within the organelle itself. As a result, chloroplast proteins are synthesized from both the nuclear and plastid genome. Plastid-encoded proteins are translated within the chloroplast, where they remain in the stroma or are imported into the thylakoid. Nuclear-encoded proteins are translated in the cytosol and have to be imported into the chloroplast. They can be sorted into one of the three membranes (the outer envelope, the inner envelope, or the thylakoid membrane) or three aqueous compartments (the inner membrane space, the stroma, or the thylakoid lumen). Nuclear-encoded proteins are imported through TOC (the Translocation to the Outer Chloroplast Envelope) and TIC (Translocation to the Inner Chloroplast Envelope) translocases, which reside on the outer and inner envelopes (Yoon et al. 2004). If they are further sorted to the thylakoid, they are imported by the SRP (Signal Recognition Particle) pathway, the Sec (Secretory) system or the Tat (Twin Arginine Translocation) system, all of which are located on the thylakoid membrane (Yoon et al. 2004). Of the thylakoid transport systems, the Tat system is unique in how it transports substrates and uses energy. The system was named after the twin arginine (RR) recognition motif that is located on the transit peptide of its own substrates. The chloroplast Tat system is made up of three membrane proteins Tha4, Hcf106, and cpTatC (TatA, TatB and TatC in E. coli) which, recognize and import substrates (Settles et al. 1997; Sargent et al. 1998; Bogsch et al. 1998). While other chloroplast translocases use nucleotide hydrolysis, the Tat system utilizes energy from the proton motive force (PMF) (Cline and Theg 2007). The mechanism that couples transport to the PMF is currently unknown. A possible mechanism mirrors the proton channel used by ATP synthase in the electron transport chain. In the proton channel, hydrogen ions protonate and deprotonate charged carboxyl-groups within the c-subunits of ATP synthase (Mitchell 1968; Rastogi and Girvin 37

1999; Cline and Theg 2007). The proton interactions induce the rotation and conformational change to produce ATP, coupling the electrochemical gradient to ATP production. There may be some charged residues on the Tat membrane proteins are involved in coupling the PMF to transport. The Tat system is able to transport fully folded proteins of various sizes (2 kDa-100 kDa and ~20Å to ~70Å) (Henry et al. 1994; Berks et al. 2003). While other translocases use chaperones to unfold their substrates, the Tat system has managed to create large enough openings to transport fully folded proteins. It does this without damaging the integrity of the membrane or dissipating the electrochemical gradient. Because of this unique feature, many studies have focused on the assembly, size and composition of the Tat pore. The two prominent models for Tat transport describe the pore as (1) forming a water-filled channel in the membrane and the other as (2) weakening the membrane through hydrophobic mismatch (Cline 2015; Gohlke et al. 2005; Cline and Theg 2007; Brüser and Sanders 2003). Stronger evidence is available for the second model, where E. coli Tat component oligomers were shown to destabilize lipid bilayers (Rodriguez et al. 2013). The oligomers, predicted in both models, are composed of TatA (bacteria). In plants and green algae, the predicted pore component is Tha4, the homologue of TatA. Tha4 (TatA) is predicted to form the translocon pore because it was found to (1) form large oligomers, (2) make contacts with substrates during the late stages of transport, (3) be in abundance over TatB and TatC, (4) associate with transport intermediates, and (5) be required for the transport step (Kostecki et al. 2010; Greene et al. 2007; Leake et al. 2008; Mori et al., 2001; Celedon and Cline, 2012; Pal et al. 2012 ;Cline and Mori, 2001). Tha4 (TatA) has a short N-terminal transmembrane domain (TMD), a hinge region that intersects the top of the bilayer and the inner core, an amphipathic helix (APH) region that rests on top of the membrane and an unstructured C-terminus tail (Settles et al. 1997; Hu et al. 2010). It has an overall “L” shape due to the perpendicular orientation of the TMD to the APH. Although oligomers of Tha4 (TatA) are predicted to make up the translocation pore, inquiries have been made about the contributions of other Tat components in forming the pore. Due to the range of substrate sizes, the other Tat components may assist Tha4 (TatA) in forming the pore. The other Tat components are TatB and TatC (Hcf106 and cpTatC in plants and green algae) (Settles et al. 1997; Sargent et al. 1998; Bogsch et al. 1998). They form the receptor complex, which recognizes the twin arginine motif on the transit peptide of substrates (Holzapfel et al. 2007; Gérard and Cline 2006). In the presence of the PMF, the bound receptor complex recruits Tha4 (TatA) oligomers to form the translocation complex (Dabney-Smith et al., 2006; Leake et al., 2008; Dabney-Smith and Cline, 2009; Kneuper et al. 2012). Hcf106 (TatB) is highly homologous to Tha4 (TatA), but has a longer C-tail (Settles et al. 1997; Hu et al. 2010). cpTatC (TatC) is a multi- spanning transmembrane protein with six TMDs. The folding pattern of the TMDs give TatC an overall “glove” shape (Ramasamy et al. 2013). Due to its homology to Tha4 (TatA), Hcf106 (TatB) was speculated to assist Tha4 (TatA) in forming the translocon pore. Earlier work studied interactions between the Tha4 and a cpTat substrate (Pal 2014) using cysteine crosslinking techniques to detect direct binding between Tha4 and the cpTat substrate. They were able to show direct binding between Tha4 and the mature domain of the cpTat substrate, the oxygen evolving complex (OE 17) at various points. In this study, the pOE17 variants (Figure 2.1, A) used previously to detect interactions with Tha4 and Hcf106 (Pal et al., 2012; Pal, 2014) were further probed for direct binding to Hcf106. The majority of the crosslinking was done with the T115C variant because of its strong association

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with Tha4. Hcf106 did not interact strongly with the pOE17-T115C variant and the results were analyzed in light of the earlier precursor-Tha4 and preliminary precursor-Hcf106 crosslinking work (Pal et al. 2012; Pal 2014). Furthermore, in this study, protease treatments and crosslinking were done to determine pOE17-T115C location upon direct binding to Hcf106. The results could show if transit peptide cleavage occurs when the substrate is stromally-exposed or membrane- protected. The results showed both the precursor and mature forms of pOE17-T115C. However, the pOE17-T115C depicted in the SDS-PAGE gel not only contained the crosslinked products (Hcf106 bound to the precursor), but un-crosslinked pOE17-T115C as well. This finding suggests that Hcf106, in addition to recognizing substrates in the receptor complex, may have a role in transporting the substrate across the membrane.

2.3 RESULTS

2.31 Oxygen Evolving Complex Crosslinked Hcf106 at the C-Proximal APH and C-tail To determine where the cpTat substrate, the oxygen evolving complex precursor (pOE17), interacts with Hcf106, we used disulfide crosslinking and transport assays. Hcf106 constructs, with cysteine substitutions in the N-terminus, APH, and TMD regions, and pOE17 constructs, with substitutions in each of its four helices, were used from previous studies (Pal, et al., 2012; Pal, 2014; Figure 2.1). (The position of the amino acids was designated based on the mature protein, after the precursor transit peptide had been enzymatically cleaved.) pOE17-T115C and pOE17- D68C and the highlighted Hcf106 variants (Figure 2.1 B) were tested in this study. Crosslinked products were formed when the Hcf106 and pOE17 variants were in close enough proximity to create a disulfide bond. Hcf106 variants were integrated into Pisum sativum thylakoid membranes. Transport assays were performed with the Hcf106-integrated thylakoids and the pOE17 variants. Crosslinked products were isolated through membrane solubilization and nickel-

Figure 2.1 VARIANTS OF OXYGEN EVOLVING COMPLEX AND HCF106

The protocol for the construction of the variants can be found in the section 2.51 of the Methods. A. Four oxygen evolving complex variants were constructed with a single cysteine point-mutation (D68C, S84C, K99C and T115C). B. Twenty Hcf106 variants were previously constructed by with a single cysteine point-mutation (four in the N-terminus, two in the transmembrane domain, twelve in the amphipathic helix domain and two in the C-terminus) and fifteen of these were used in crosslinking assays. (Chimera space-filling model adapted from the Protein Databank, http://www.rcsb.org/3d- view/2MI2/1).

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affinity purification as described in the Methods (Section 2.5). pOE17 variants were constructed with a 6His-tag to bind the column. Unbound Hcf106 variants were found in the supernatant and bound fractions were found in the elution fraction (Section 2.3). Bound and unbound fractions were visualized using SDS-PAGE and fluorography. Crosslinked products were detected by the tritiated Hcf106 variants in the elution fraction. Un-crosslinked Hcf106 were detected in the supernatant. The pOE17-T115C variant did not crosslink with the N-terminus (residues 6-10), TMD region (residues 13-14), APH region (residues 31-64) or C-terminus (residues 67-128) of the tested Hcf106 residues (Figure 2.3). However, Pal (2014) demonstrated interaction between pOE17- T115C and A128C in the C-tail of Hcf106 (Table 2.1). The pOE17-D68C variant crosslinked with one residue (S53) in the APH region (Figure 2.2). The Hcf106-S53C band showed up at 26kDa. Prior work also demonstrated strong interaction with the pOE17-D68C variant in the C-tail and at the end of the APH region (Pal 2014). Weak or no interaction was also seen in the N-terminus and most of the APH region

Figure 2.2 HCF106 CROSSLINKING FOR OE17-D68C AND –T115C

A. Tritiated pOE17-D68C was transported into thylakoids with variant Hcf106 (-G6C, -S53C, and -S58C) in lanes 1- 3. Crosslinking between pOE17-K99C and Hcf106-D67C was used as a positive control in lane 4 (+). Crosslinked

products were detected through the tritiated Hcf106, visualized on the SDS-PAGE gel. Crosslinking was detected between the pOE17-D68C and Hcf106-S53C B. Tritiated pOE17-T115C was transported into thylakoids with one of fifteen Hcf106 variants in lanes 1-24. Crosslinking between pOE17-K99C and Hcf106-I64C and Hcf106-E55C were used as positive controls in lanes 4, 8, 12, 16, 20, and 24 (+). Crosslinked products were detected through the tritiated Hcf106 and visualized on the SDS-PAGE gel. pOE17-T115C formed weak crosslinks only with Hcf106-S58C and

Hcf106-I64C, which correspond to the C-proximal APH region.

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Table 2.1 INTEGRATED TABLE OF CROSSLINKING BETWEEN pOE17 AND HCF106 VARIANTS

Integrated table combining crosslinking results from Pal et al. (2014) and this study (*). pOE17-D68C and -K99C had weak interactions with the N-terminus of Hcf106 and none of the pOE17 variants interacted with the TMD region. pOE17-D68C and - K99C had weak interactions with several residues in the APH region. pOE17-D68C, -S84C, and -K99C had strong interactions with the C-proximal APH region (S53C-I64C). pOE17-D68C and -S84C had strong interactions in the C-tail and pOE17-K99C had a strong interaction with one residue in the C-tail (A129C).

Hcf106 Variants Oxygen Evolving Complex Variants pOE17-D68C* pOE17-S84C pOE17-T115C* pOE17-K99C N-Terminus G6C –* – –* +/– V7C +/– – – +/– G8C +/– n.d. –* +/– P10C – – –* – Transmembrane Domain L13C – – –* – V14C – – –* – Amphipathic Helix Region V31C – – –* – R33C +/– – –* – N34C – – –* +/– G36C – – –* – T38C – – –* – L39C +/– – –* – E48C – – –* +/– S53C +* – –* +/– E55C + + – + S58C –* + +/–* + F61C + n.d. – + I64C + + +/–* + C-Terminus D67C +* + n.d. n.d. A128C + + – +

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2.32 Hcf16 Crosslinked to The Precursor and Mature Protein of OE17-T115C Under Transport Conditions To determine whether the pOE17 substrate can be transported while crosslinked to Hcf106, we performed transport assays with thermolysin treatments. The thermolysin treatments were used to reveal whether the substrate is membrane protected (transported) or stromally-exposed when crosslinked to Hcf106. The Hcf106-G6C and Hcf106-I64C variants were tested with the pOE17- T115C variant. Hcf106 integration, the transport assay, membrane solubilization and nickel- affinity purification were carried out as described in the Methods (Section 2.5). For this experiment and subsequent to transport, half of the samples were treated with thermolysin and half were treated with deionized water (untreated samples). Crosslinked products with membrane protected substrates (transported) would not be exposed to proteolysis, while substrates outside of the membrane would be degraded. To visualize the substrates, the substrate was tritiated and the samples were subject to analysis by polyacrylamide gel electrophoresis (SDS-PAGE). The pOE17-T115C substrate was transported through the Hcf106-G6C and Hcf106-I64C -integrated thylakoids (Figure 2.4). In untreated samples, the precursor and mature domain of OE17-T115C is present. In the treated samples, the precursor is absent and the mature is present. This demonstrated that the precursor was degraded by thermolysin (Figure 2.3).

Figure 2.3 CROSSLINKING AND THERMOLYSIN ASSAY FOR pOE17-T115C AND HCF106-G6C AND –I64C

pOE17-T115C was transported into tritiated Hcf106-G6C and –I64C integrated thylakoids, visualized on the SDS-PAGE gel, in lanes 1-6. Crosslinking between pOE17-K99C and Hcf106-E55C was used as a positive control (+). Subsequent to crosslinking and transport, thermolysin was added to the samples (total concentration 0.1mg/ml) in lanes 2, 4, and 6. Untreated samples (1, 3, and 5) were treated with the same amount of deionized water. The precursor and mature domain of OE17-T115C were present in the untreated samples and in the treated samples, the precursor was absent from proteolytic degradation and the mature protein is present.

2.4 DISCUSSION

2.41 Hcf106 Integrated Interaction Map The aim of this study was to investigate whether Hcf106, along with Tha4, make up the composition of the cpTat translocon pore. Tha4 is predicted to form the translocon pore because of its abundance over TatB and TatC, large oligomers, and because it is required for the transport step. In a previous study, direct interaction between Tha4 and the oxygen evolving complex precursor (pOE17) was shown using cysteine crosslinking (Pal 2014). Since the pore can accommodate large substrates (2 nm-7 nm), other cpTat proteins were hypothesized to constitute the pore with Tha4. Hcf106 was investigated as a potential pore component because of its homology to Tha4 and demonstrated direct interaction with pOE17 using cysteine crosslinking (Pal 2014). Four cysteine variants of pOE17 (pOE17-D68C, -S84C, -K99C, and -T115C) were 42

constructed for the crosslinking assays and an interaction map for pOE17 and Hcf106 was constructed, in which the remaining assays, in this study for pOE17-D68C and -T115C, were integrated (Table 2.1). Interactions between pOE17-K99C and Hcf106-E55C or –I64C were used as positive controls (Figure 2.2). Previous work demonstrated strong interactions between pOE17-K99C and the two Hcf106 variants. pOE17-T115C only formed crosslinks with Hcf106-S58C and -I64C, and pOE17-D68C formed crosslinks with Hcf106-S53C, not Hcf106-S58C. Table 2.1 integrated our results with Pal (2014), helping to complete the interaction map for pOE17 and Hcf106. Table 2.1 showed weak interactions in the N-terminus for pOE17-D68C and –K99C and no interactions for any variant in the TMD. A few weak interactions in the N-proximal APH region were found for pOE17-D68C and –K99C. Towards the C-proximal end of the APH, strong interactions were seen with pOE17- D68C, -S84C and –K99C (and one for –T115C). Excluding non-determined (n.d.) assays from Table 2.1, interactions in the C-tail were strong for pOE17–D68C, –S84C and –K99C. Together, the results show that the C-proximal APH and C-tail residues are more important for substrate binding. This contrasts with the Tha4 interaction map, where strong interactions were observed within all domains, including the N-terminus, for pOE17-D68C (Pal et al. 2014). The assays for the other pOE17 variants (-D59C, -S84C, and -T115C) are mostly non-determined in the APH region of Tha4 and in some of the N-terminus. There were strong interactions for one residue in the N-terminus for pOE17-D59C (Tha4-P9C) and for pOE17-T115C (Tha4-G5C). There were also strong interactions in the C-terminal APH region of Tha4 (-F48C, -T50C, and -K53C) for all the pOE17 variants except pOE17-S84C, which had strong interactions for only two residues in that region (-T50C and -K53C). Together, the two interaction maps suggest a binding preference on the four-helix bundle of pOE17 (Figure 2.1). The T115C substitution formed strong crosslinks in the C-terminal APH region of Tha4 and one strong crosslink in the N-terminus. In contrast, the T115C substitution only crosslinked with two Hcf106 variants (Hcf106-S58C and –I64C) in the C-proximal APH region. The helix with the S84C substitution formed one strong crosslink in the N-proximal APH and in the C-proximal APH region of Tha4. Weak crosslinks were found in the mid-APH region, and a weak crosslink was found in the C-tail and in the N-terminus. For the Hcf106 interaction map, the S84C substitution formed strong crosslinks in the C-proximal APH region and the C-tail. The helix with the D68C substitution formed strong crosslinks throughout the regions of Tha4, and formed weak crosslinks in the N-terminus and strong crosslinks in the C-proximal APH and C-tail of Hcf106. The interaction maps may suggest that Hcf106 interacts with three of the pOE17 helices (pOE17-D68C, -S84C, and K99C) at the start of transport, where, in the stroma, the C-tail and APH regions of Hcf106 are accessible. Tha4 may interact with pOE17 throughout its transport to the lumen, at the C-tail, APH, and N-terminus (pOE17-D68C, -S84C, and T115C). While important for transport, it is unclear whether Hcf106 is a pore component or stabilizes and transfers the substrate for transport. Our results point towards a “hand-off” (Pal et al. 2014) and stabilization model where Hcf106 stabilizes the precursor at the mature domain during Tha4 oligomerization and then transfers it to Tha4 for transport. For stabilization, in the stroma, Hcf106 could bind the mature domain of the precursor at the C-tail and C-proximal APH region, shown by the crosslinking data in Table 2.1. Hcf106 could then transfer it to the Tha4 oligomers for transport, the precursor interacting with all domains except the TMD and C-tail, which is shown in the Tha4 crosslinking interaction map in Pal et al. (2014) for the pOE17-D68C variant. In all, the precursor could interact strongly with the stroma-exposed domains of Hcf106 and Tha4 the (C-

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tail and APH domains), and, upon transport, the membrane-protected domains of Tha4 (the N- terminus). While the “hand-off” model is plausible, dual-crosslinking data for Hcf106 variants did not show more co-precipitation of Hcf106 at the start of transport (Pal, 2014), which would be expected if Hcf106 was transferring the precursor to Tha4. Hcf106 may just have a role in stabilizing the precursor, in addition to its role in the receptor complex.

2.42 Combination of Crosslinked & Un-crosslinked OE17T115C Recovered In this study, crosslinking, transport assays, and thermolysin treatments were performed to determine if OE17 maintains contact with Hcf106 during transport. The protease activity of thermolysin degrades untransported precursors (exposed to the stroma) and has no effect on transported ones (membrane-protected). A tritiated pOE17 variant ([3H] pOE17-T115) was crosslinked to non-radioactive Hcf106 variants (Hcf106-G6C and Hcf106-I64) under transport conditions. The individual assays were divided into an untreated sample, containing deionized water, and a treated sample containing thermolysin. Following membrane solubilization, a nickel- affinity column was used to isolate crosslinked products and SDS-PAGE/fluorography was used to visualize the results. Transported precursors (membrane-protected) were expected to be present in the treated and untreated samples, while untransported precursors were expected to be present in the untreated sample and degraded by thermolysin in the treated sample. pOE17- T115C and Hcf106-G6C were not expected to crosslink based on previous assays (Figure 2.3, Table 2.1). However, pOE17-T115C was expected to crosslink with Hcf106-I64C (Figure 2.3, Table 2.1). For Hcf106-G6C and Hcf106-164C, the precursor and the mature protein were present in the untreated samples. In the treated samples, the precursor was degraded by thermolysin while the mature protein was unaffected. The presence of OE17-T115C in the Hcf106-G6C sample did not correspond with the interaction map, described in Table 2.1. The bands present in the Hcf106- G6C untreated column (Figure 2.4) would have to be un-crosslinked OE17-T11C, including both precursor and mature proteins that were transported across the membrane. The bands in the Hcf106-I64C untreated column are likely a mixture of crosslinked and un-crosslinked OE17-T11C, since pOE17-T115C and Hcf106-I64C crosslinked in previous assays (Figure 2.4, Table 2.1). The nickel-affinity column, instead of being selective to crosslinked precursors and mature proteins, pulled down all available OE17-T115C. The experiment could be revised in two ways. (1) Instead of pulling down OE17, a FLAG-affinity column could be used to pull down all Hcf106 variants which would be crosslinked to tritiated OE17. Any un-crosslinked Hcf106 would not be visible on SDS-PAGE due to its lack of a radiolabel. (2) The assay protocol could remain same with a slight modification to the SDS-PAGE sample buffer. 2-Mercaptoethanol (BME) could be removed from the sample buffer, so the crosslinks would not be reduced. Crosslinked products would then show up at a higher molecular weight, differentiating it from un-crosslinked precursors. Results from revised versions of the assays could give more information about the role of Hcf106 in transport. If OE17 can be transported while crosslinked to Hcf106, it’s possible Hcf106 may be a part of the translocon pore, similar to Tha4, or may have a role stabilizing the substrate during luminal processing. If OE17 can be transported while crosslinked to Hcf106, it is most likely to be crosslinked to a residue in the N-terminus or N-terminal APH region that contacts the membrane. Topological changes were detected in Hcf106 upon precursor binding, where the N-proximal APH region tilted into the membrane (Zhang 2015). In addition to previously mentioned corrections, the revised assays would incorporate Hcf107 variants in the N-proximal APH region (Hcf106- R33C). 44

2.43 cpTat Transport Model: Membrane Weakening It is possible that the cpTat system doesn’t use a water-filled pore to transport substrates. Computer simulated studies demonstrated how the TMD of Tha4 (TatA) oligomers could cause membrane disruption through hydrophobic mismatching (Rodriguez et al. 2013). Specifically, the short TMD was shown to cause lipid disordering and membrane thinning (Rodriguez et al. 2013). Membrane weakening by TatA was also shown in E. coil (Hou et al. 2018). In the resting state, the N-proximal APH is tilted into the membrane, preventing hydrophobic mismatching by the short TMD. Upon substrate binding, the N-proximal APH becomes more parallel with the membrane, pulling the TMD towards the surface of the membrane. Without the counteraction of the tilted APH, the short TMD caused hydrophobic mismatching and membrane thinning. The destabilization of the membrane was measured by proton leakage. Both studies demonstrated how substrate-induced conformational change in TatA destabilizes the membrane. The membrane weakening model has been proposed in other studies that focus on Hcf106. Because Hcf106 and Tha4 are homologous, conformational changes in Hcf106 are suspected to cause membrane destabilization similar to Tha4. In two studies, substrate-induced conformational changes in Hcf106 supported the membrane weakening model (Habtemichael, 2017; Zhang et al. 2015). Similar to Rodriguez et al. and Hou et al., the N-proximal APH region and the TMD were mostly affected upon substrate-binding (Habtemichael et al. 2017; Zhang et al. 2015). Both studies used cysteine accessibility labeling technique to detect these conformational changes. In one study, the N-proximal APH angled into the membrane upon substrate-binding, where it became less accessible to the stroma (Habtemichael et al. 2017). In contrast, the other study found that the N-proximal end of the APH became more exposed to the stroma and more parallel with the membrane upon substrate-binding (Zhang et al. 2015). The topology changes from Zhang et al. are similar to those found in Hou et al. for Tha4. It’s possible that the N-proximal APH region of Hcf106 prevents hydrophobic mismatch between the short TMD and the lipid bilayer. Upon binding, the removal of the N-proximal APH region could cause the membrane to compress to match the short hydrophobic region of the TMD. The aggregate of TMD’s from Hcf106 and the Tha4 oligomer could compress the bilayer enough to transport the substrate. The cpTat system is able (1) to transport folded substrates, of various sizes, (2) without disrupting the membrane, (3) using the proton motive force. “Membrane weakening” is a more suitable transport model for the cpTat system because it explains how cpTat transports substrates of various sizes without dissipating the PMF. When the substrate binds the receptor complex (cpTatC-Hcf106), in the presence of the PMF, Tha4 oligomers are recruited together to form the translocase. The combined hydrophobic mismatch of Tha4 (Rodriguez et al. 2013) and Hcf106 TMDs, compress the membrane and create a transient opening around the substrate. The TMDs of Tha4 and Hcf106 are essential in order to compress and weaken the membrane for the substrate to transport through. Membrane weakening may explain why the PMF is not dissipated during transport. Dissociation of substrate allows Hcf106 and Tha4 to quickly assume their resting-state conformation where the N-proximal APH region is tilted into the pore. (Simulations from Rodriguez et al. also suggest that Tha4 disassembles rapidly following transport.) The resting-state conformation would decompress the membrane and close the opening. Although ~80,000 protons are released from the pH gradient during each translocation event, only ~10% of those protons are not coupled to protein transport (Alder and Theg 2003). A transient opening would explain why there is little proton leakage and the PMF is not dissipated. Future studies about membrane weakening would

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focus on whether the N-terminus of Hcf106 can destabilize the membrane similar to Tha4 (Hou et al. 2018). Future studies about the cpTat transport event would focus on (1) identifying the motor that could pull the substrate across the membrane and (2) investigating how the PMF is coupled to transport.

Figure 2.4 CHLOROPLAST TWIN ARGININE TRANSLOCATION PATHWAY MEMBRANE WEAKENING TRANSPORT MODEL

Precursor-induced conformational change causes the tilted APH regions of Hcf106 and Tha4 (Panel A.) to become parallel with the bilayer (Panel B. and C.). The conformational change pulls the short TMD of Hcf106 and Tha4 towards the stromal face of the bilayer. To resolve the discrepancy between the thickness of the bilayer and the short TMDs of Hcf106 and Tha4, the membrane becomes compressed (Panel C.). A transient opening is created by the compression, which allows the precursor to be transported into the thylakoid lumen (Panel D.).

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2.5 METHODS

2.51 IN VITRO SYNTHESIS OF HCF106 AND TAT SUBSTRATE CONSTRUCTS Twenty Hcf106 constructs (Hcf106XnC) were generated with cysteine substitutions, designated by the amino acid abbreviation (X), the residue number (n) and the abbreviation for cysteine (C). The coding sequence for the mature Hcf106 was used as a template for the constructs. The beginning of the sequence is as follows: MASLFGVGAPEA. The QuikChange Mutagenesis protocol from Agilent Technologies was utilized to generate the constructs. DNA sequencing was performed to verify the cysteine substitutions in the constructs in the Center for Bioinformatics and Functional Genomics at Miami University. (Figure 2.1 B) Four Oxygen Evolving complex precursor constructs (pOE17XnC) were generated with cysteine substitutions. A modified form of the precursor, consisting of a C-terminal polylinker and histidine- tag, was used as the template for the constructs. The polylinker consisted of three repeats of four glycines and one serine [(G4S)3], and the Histidine-tag (His6) was constructed at the end of the C- terminus. This template was a generous gift from Dr. Kenneth Cline from the University of Florida. The QuikChange Mutagenesis kit from Stratagene was utilized to generate the four constructs (Asp68, Ser84, Lys99, and Thr115) with PCR. The position of the amino acids was designated based on the mature protein, after the precursor transit peptide had been enzymatically cleaved. DNA sequencing was performed to verify the cysteine substitutions in the constructs in the Center for Bioinformatics and Functional Genomics at Miami University. (Figure 2.1 A)

2.52 ISOLATION OF CHLOROPLASTS AND THYLAKOIDS (All samples were kept on ice except during resuspension). Intact chloroplasts were isolated from 10-12-day old Little Marvel pea (Pisum sativum) seedlings using the protocol from Cline et al (Cline et al., 1986). The chloroplasts were resuspended (from previous pelleting) in IB (import buffer: 50 mM HEPES-KOH, pH 8.0, and 330 mM sorbitol) to 1mg/mL chlorophyll. Thylakoids were isolated by osmotic lysis of intact chloroplasts at 2mg/mL chlorophyll in 10 mM HEPES-

KOH, pH 8.0, and 10 mM MgCl2 (Mori and Cline,1998). Thylakoids were separated from the lysate by pelleting (3,200 g, 8 min) and washed with a resuspension in IB with 10mM MgCl2. To complete the wash, the resuspended thylakoids were pelleted and the supernatant was discarded. Washed thylakoids were resuspended in IBM (1X IB and 10mM MgCL2) to 1 mg/ml chlorophyll.

2.53 PRECURSOR RADIOLABEL-INCORPORATED TRANSLATION All Hcf106 and precursor variants were generated by in vitro translation. This was carried out in a wheat germ extract, with capped mRNA and tritiated [3H] Leucine (Dabney-Smith et al., 2006). All products were diluted in a 1:1 ratio with 60mM leucine 2X IB (import buffer) after translation.

2.54 PRECURSOR INCUBATION WITH DTNB To prevent precursor-precursor crosslinking, the precursor translation product was incubated with 80 µM DTNB (2- nitrobenzoic acid) for 30 min at room temperature (Pal et al 2013). Following the incubation, the translation product was placed on ice.

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2.55 HCF106 INTEGRATION INTO THYLAKOIDS In vitro translated [3H] Hcf106XnC was integrated into the membranes of isolated thylakoids. A 1:1:2 ratio of [3H] Hcf106XnC, isolated thylakoids, and IBM, with 1.5 mM DTT at final concentration, was incubated at 25°C for 25 min. Integrated thylakoids were pelleted, washed with IBM (500 µl) and resuspended in IBM to the total volume desired for the transport assay.

2.56 TRANSPORT ASSAY The DTNB-treated precursor, integrated thylakoids and IMB were combined in a 2:2:1 ratio. The transport sample was incubated for 25 min at 25°C in the presence of 150 µmol.m-2.s-1 light. For the thermolysin treatment, transport samples were split into two samples such that there were duplicates of the [3H] Hcf106XnC thylakoid-integrated samples. Cold IBM (500 µl) was added to the samples on ice to arrest transport. The samples were pelleted and resuspended in IBM (150 µl) (Gérard and Cline, 2006).

2.57 THERMOLYSIN TREATMENT Half of the transport [3H] Hcf106XnC duplicates were treated with thermolysin at a total concentration of 0.1mg/ml. The other duplicates were treated with an equal volume of deionized water. All samples were incubated at 4°C for 30 min. Ethylenediaminetetraacetic acid (EDTA) was added to the samples, at a final concentration of 5 mM, to arrest the protease activity of thermolysin. Samples were pelleted and resuspended in 5 mM EDTA, 1X IB to their original volume after the transport reaction was arrested (150 µl). Samples were pelleted again and resuspended in 1X IB (150 µl total volume).

2.58 MEMBRANE SOLUBILIZATION To solubilize the membrane, thermolysin treated samples (including water control samples) were pelleted and resuspended in tris-buffered saline (TBS: 50 mM Tris-HCl, pH 7.5, 500 mM NaCl), to a total volume of 22 µl. Equal amount of 2% SDS was added and the samples were incubated at 37°C for 10 min. Samples were centrifuged at 13.4 rpm for 6 minutes at 4°C. The supernatant was transferred to the nickel-affinity column.

2.59 NICKEL-AFFINITY PULL-DOWN The nickel-affinity resin was prepared with equal volumes of sample and pre-charged IMAC resin (GE Healthcare) with 500 µl of binding buffer (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 5 mM imidazole, 1% Triton X-100, 0.5% deoxycholate). The supernatant produced at the end of the membrane solubilization protocol was added to the nickel resin. The samples were incubated at 4°C, end-over-end (360° vertical rotation) overnight (~16 hrs). The nickel beads, bound to the crosslinked products, were pelleted at 500 g for 2 minutes. A sample of the unbound [H3] Hcf106XnC was taken from the supernatant for SDS-PAGE (20 µl). The nickel beads were washed and pelleted (500 g, 2 min) twice, using 1 ml of wash buffer (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 20 mM imidazole, 1% Triton X-100, 0.5% deoxycholate) and once with 1 ml of the final wash buffer (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 20 mM imidazole, 0.05% 93 Triton X-100). After pelleting (500 g, 2 min), 50 µl of the elution buffer (20 mM Tris-HCl, pH 6.8, 0.5 M NaCl, 0.5 M imidazole, 6 M urea, 5% SDS) was added to the samples. The samples were incubated at 4°C overnight (~16 hrs). The samples were pelleted (13.4 rpm, room temperature, 5 min) and the supernatant was removed for SDS-PAGE.

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2.510 CROSSLINK-PRODUCT DETECTION Samples were analyzed by SDS-PAGE and fluorography. Supernatant (unbound fractions) and elution samples were diluted with 2X SSB (SDS-PAGE Sample Buffer, Cold Springs Harbor) in a 1:1 ratio and the samples were run on SDS-PAGE. For fluorography analysis, the gels were treated with DMSO for 25 min, PPO/DMSO for 45 min, and washed with DI water for 30 min. The gel was dried, exposed for five days at -80°C and then developed in a dark room using manual dip tanks.

2.6 REFERENCES Alder, N.N. and Theg, S.M. 2003. Energetics of protein transport across biological membranes: a study of the thylakoid ΔpH-dependent/cpTat pathway. Cell. 112(2):231-242. Berks, B.C., Palmer, T. and Sargent, F. 2003. The Tat protein translocation pathway and its role in microbial physiology. Adv. Microb. Physiol. (47)187-254. Bogsch, E.G., Sargent, F., Stanley, N.R., Berks, B.C., Robinson, C. and Palmer, T. 1998. An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria. J. Biol. Chem. 273(29):18003-18006. Brüser T., Sanders C. 2003. An alternative model of the twin arginine translocation system. Microbiol. Res. 158(1):7–17. Celedon, J.M. and Cline, K. 2012. Stoichiometry for binding and transport by the twin arginine translocation system. J. Cell. Biol. 197(4):523-534. Cline, K. 2015. Mechanistic aspects of folded protein transport by the twin arginine translocase (Tat). J. Biol. Chem. 290(27):16530-16538. Cline, K. and Mori, H. 2001. Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC– Hcf106 complex before Tha4-dependent transport. J. Cell. Biol. 154(4):719-730. Cline, K., Theg, S.M. 2007. The Enzymes: Molecular Machines Involved in Protein Transport across Cellular Membranes. In: Dalbey, R., Koehler, C., Tamanoi, F. editors. The Sec and Tat protein translocation pathways in chloroplasts. Molecular Machines Involved in Protein Transport across Cellular Membranes. London(UK):Academic Press. 25 p. 463-492. Dabney-Smith, C., and Cline, K. 2009. Clustering of C-terminal stromal domains of tha4 homo- oligomers during translocation by the tat protein transport system. Mol. Biol. Cell. 20(7):2060– 2069. Gérard, F. and Cline, K. 2006. Efficient twin arginine translocation (Tat) pathway transport of a precursor protein covalently anchored to its initial cpTatC binding site. J. Biol. Chem. 281(10):6130-6135. Gohlke, U., Pullan, L., McDevitt, C.A., Porcelli, I., de Leeuw, E., Palmer, T., Saibil, H.R. and Berks, B.C. 2005. The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter. Proc. Natl. Acad. Sci. USA. 102(30):10482–10486. Greene, N.P., Porcelli, I., Buchanan, G., Hicks, M.G., Schermann, S.M., Palmer, T. and Berks, B.C. 2007. Cysteine scanning mutagenesis and disulfide mapping studies of the TatA component of the bacterial twin arginine translocase. J. Biol. Chem. 282(33):23937-23945. 49

Habtemichael, A.G. 2017. Insights into the Chloroplast Tat Mechanism of Transport [Doctoral dissertation]. [Oxford (OH)]: Miami University. Henry, R., Kapazoglou, A., McCaffery, M. and Cline, K. 1994. Differences between lumen targeting domains of chloroplast transit peptides determine pathway specificity for thylakoid transport. J. Biol. Chem. 269(14):10189-10192. Holzapfel, E., Eisner, G., Alami, M., Barrett, C.M., Buchanan, G., Lüke, I., Betton, J.M., Robinson, C., Palmer, T., Moser, M. et al. 2007. The entire N-terminal half of TatC is involved in twin-arginine precursor binding. Biochemistry. 46(10):2892-2898. Hou, B., Heidrich, E.S., Mehner-Breitfeld, D. and Brüser, T. 2018. The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding. J. Biol. Chem. .jbc-RA118. Hu, Y., Zhao, E., Li, H., Xia, B. and Jin, C. 2010. Solution NMR structure of the TatA component of the twin-arginine protein transport system from gram-positive bacterium Bacillus subtilis. J. Am. Chem. Soc. 132(45):15942-15944. Kneuper, H., Maldonado, B., Jäger, F., Krehenbrink, M., Buchanan, G., Keller, R., Müller, M., Berks, B.C. and Palmer, T. 2012. Molecular dissection of TatC defines critical regions essential for protein transport and a TatB–TatC contact site. Mol. Microbiol. 85(5):945-961. Kostecki, J.S., Li, H., Turner, R.J. and DeLisa, M.P. 2010. Visualizing interactions along the Escherichia coli twin-arginine translocation pathway using protein fragment complementation. PLoS One. 5(2):e9225. Leake, M.C., Greene, N.P., Godun, R.M., Granjon, T., Buchanan, G., Chen, S., Berry, R.M., Palmer, T. and Berks, B.C. 2008. Variable stoichiometry of the TatA component of the twin- arginine protein transport system observed by in vivo single-molecule imaging. Proc. Natl. Acad. Sci. USA. 105(40):15376-15381. Mitchell, P. 1968. Chemiosmotic Coupling and Energy Transduction. FEBS. Lett. 87:171-179. Mori H., Summer E.J., Cline K. 2001. Chloroplast TatC plays a direct role in thylakoid (Delta)pH- dependent protein transport. FEBS. Lett. 501(1):65–68. Pal, D. 2014. Mapping of precursor mature domain binding site on cpTat receptor complex component Hcf106. [Doctoral dissertation]. [Oxford (OH)]: Miami University. Pal, D., Fite, K., Dabney-Smith, C. 2013. Direct interaction between a precursor mature domain and transport component Tha4 during twin arginine transport of chloroplasts. Plant Physiol. 161(2):990-1001. Pal, D., Fite, K. and Dabney-Smith, C. 2012. Direct Interaction between Precursor Mature Domain and Transport Component Tha4 during Twin Arginine Transport (Tat) of Chloroplasts. Plant. Physiol. pp-112.Pal, D. 2014. Mapping of precursor mature domain binding site on cpTat receptor complex component Hcf106. [Doctoral dissertation]. [Oxford (OH)]: Miami University. Ramasamy, S., Abrol, R., Suloway, C.J. and Clemons, W.M. 2013. The glove-like structure of the conserved membrane protein TatC provides insight into signal sequence recognition in twin- arginine translocation. Structure. 21(5):777-788.

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Rastogi, V. K., Girvin, M.E. 1999. Structural changes linked to proton translocation by subunit c of the ATP synthase. Nature. 402(6759):263. Rodriguez, F., Rouse, S.L., Tait, C.E., Harmer, J., De Riso, A., Timmel, C.R., Sansom, M.S., Berks, B.C. and Schnell, J.R. 2013. Structural model for the protein-translocating element of the twin-arginine transport system. Proc. Nat. Acad. Sci. 110(12):e1092-E1101. Rollauer, S.E., Tarry, M.J., Graham, J.E., Jääskeläinen, M., Jäger, F., Johnson, S., Krehenbrink, M., Liu, S.M., Lukey, M.J., Marcoux, J., McDowell, M.A. 2012. Structure of the TatC core of the twin-arginine protein transport system. Nature. 492(7428):210-214. Sargent, F., Bogsch, E.G., Stanley, N.R., Wexler, M., Robinson, C., Berks, B.C. and Palmer, T. 1998. Overlapping functions of components of a bacterial Sec‐independent protein export pathway. EMBO. J. 17(13):3640-3650. Settles, A.M., Yonetani, A., Baron, A., Bush, D.R., Cline, K. and Martienssen, R. 1997. Sec- independent protein translocation by the maize Hcf106 protein. Science. 278(5342):1467-1470. Yoon, H. S., Hackett, J. D., Ciniglia, C., Pinto, G., Bhattacharya, D. 2004. A molecular timeline for the origin of photosynthetic eukaryotes. Mol. Biol. Evol. 21(5):809-818. Zhang, L. 2015. Biophysical and biochemical investigation of the structure of chloroplast twin arginine transport component Hcf106 [Doctoral dissertation]. [Oxford (OH)]: Miami University. Zhang, L., Liu, L., Maltsev, S., Lorigan, G.A., Dabney-Smith, C. 2014. Investigating the interaction between peptides of the amphipathic helix of Hcf106 and the phospholipid bilayer by solid-state NMR spectroscopy. Biochim. Biophys. Acta. 1838(1):413-418. Zhang, L., Liu, L., Maltsev, S., Lorigan, G.A., Dabney-Smith, C. 2013. Solid-state NMR investigations of peptide–lipid interactions of the transmembrane domain of a plant-derived protein, Hcf106. Chem. Phys. Lipids. 175:123-130.

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Chapter 3. CONCLUSIONS

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2.7 CONCLUSION The composition of the chloroplast Twin Arginine Translocation (cpTat) translocon pore was investigated in this study. The cpTat system is important for energy production, respiratory and photosynthetic symbiosis and pathogenesis in plants and animals. It is an ancestral translocase (Berks 1996; Dilks et al. 2003) that can be found in plant and algal chloroplasts and in some archaea and bacterial mitochondria (Cline and Theg 2007). The Tat system is able to transport fully folded proteins, using the proton motive force (Berks et al. 2000; Rodrigue et al. 1999; DeLisa et al. 2003) and relies on the electrochemical gradient to power transport of substrates (Cline and Theg 2007). It is able to accomplish this without damaging the integrity of the membrane and dissipating the electrochemical gradient or proton motive force (PMF). The cpTat system uses three membrane proteins, Tha4, Hcf106, and cpTatC (Settles et al. 1997; Sargent et al. 1998; Bogsch et al. 1998), in a cyclical PMF-dependent process, to transport substrates by creating a temporary pore in the membrane (Mori et al. 1999; Mori et al. 2001; Fincher et al. 2003). Hcf106 and Tha4 are homologous single-spanning membrane proteins, with a short N- terminal transmembrane domain (TMD), a hinge region at the top of the bilayer, an amphipathic helix (APH) region on top of the membrane and an unstructured C-terminus tail (Settles et al. 1997; Hu et al. 2010). cpTatC is a multi-spanning membrane protein with six transmembrane domains (TMDs) and has both of its termini on the cis side of the membrane. While Hcf106 and Tha4 have an “L” shape with the TMD and APH perpendicular to each other, cpTatC takes on the shape of a “glove” due to the folding pattern of its TMDs (Ramasamy et al. 2013). Tha4 is predicted to form the translocon pore because it forms large oligomers, is abundant over Hcf106 and cpTatC, interacts directly with the mature domain of precursors and is a requirement for the transport (Kostecki et al. 2010; Greene et al. 2007; Leake et al. 2008; Mori et al., 2001; Celedon and Cline, 2012; Pal et al. 2012; Cline and Mori, 2001).

Because the cpTat system is able to accommodate substrates that vary in size, Hcf106 was investigated as another component of the translocation pore. Hcf106 was investigated because of its structural similarity to Tha4 and direct binding between it and the mature domain of a cpTat substrate that was demonstrated (Pal, 2014) using cysteine-crosslinking. In addition to recognizing substrates in the receptor complex, Hcf106 may have a role in transporting the substrate across the membrane. In this study, the cysteine-crosslinking assays were continued with variants of Hcf106 and the Oxygen Evolving Complex precursor (pOE17). There are four substrate variants (pOE17-D68C, -S84, -K99C, and -T115C) and twenty Hcf106 variants on the C-terminus, APH, TMD, and N-terminus. Direct binding was shown for three pOE17 variants (- D68C, -S84, and -K99C) in the N-terminus, C-terminal APH region, and the C-terminus of Hcf106 (Pal et al. 2014). In this study, cysteine-crosslinking was performed between the Hcf106 variants and pOE17-T115C and –D68C. pOE17-T115C only formed crosslinks with two Hcf106 variants in the C-proximal APH region (Hcf106-S58C and -I64C) and pOE17-D68C formed a strong crosslink in the C-proximal APH region. Overall, the integrated data (Table 2.1) showed that the C-proximal APH and C-tail of Hcf106 had the strongest interactions with pOE17, and a comparison of the Tha4 (Pal et al 2014) and Hcf106 interaction maps suggested binding preferences for the two membrane proteins on the four-helix bundle of pOE17.

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Because of its ability to bind the precursor mature domain, Hcf106 may be a part of the translocation pore. Another possibility is that it stabilizes the precursor during Tha4 assembly and later transfers it for transport, the “hand-off” mechanism. Comparing the pOE17-D68C crosslinking data for Tha4 and Hcf106, pOE17-D68C interacts strongly with the N-terminus and APH domains of Tha4. However, interactions with Hcf106 were weak or nonexistent in the N- terminus and strong in the C-tail end of the APH domain. If Hcf106 were a part of the pore, the mature domain would be in close proximity to the N-terminus and there would have been stronger binding to those residues. It is possible that the strong interactions at the C-tail end of the APH domain of Hcf106 support the “hand-off” mechanism where the mature domain interacts with Hcf106 in the APH, is transferred to the APH region of Tha4, and then transported where it interacts with the N-terminus of Tha4. Crosslinking assays need to be done for the other pOE17 variants (-D68C, -S84, and -T115C) to determine if interactions are also weak in the N-terminus and stronger in the APH domain of Tha4.

Lastly, this study addressed whether cpTat substrates stay in contact with Hcf106 during transport. Protease treatments and crosslinking assays between Hcf106 and pOE17 were performed to determine whether substrates crosslinked to Hcf106 could be transported. Thermolysin was used to degrade stromally-exposed precursors that were crosslinked to Hcfc106, having no effect on membrane-protected (transported) ones. However, crosslinked products and uncrosslinked OE17 were indistinguishable from each other following the assay. The presence of precursor in the Hcf106-G6C crosslinking assay did not correspond with the interaction map for pOE17-T115 (Table 2.1), where pOE17-T115 did not crosslink with Hcf106- G6C (Figure 2.3, A). The bands from the Hcf106-G6C assay (Figure 2.4) would have to be uncrosslinked pOE17-T115C. It was unclear whether the pOE17, pulled-down from a nickel- affinity column and visualized on the gel, were un-crosslinked or crosslinked to Hcf106-T115C. The assay could be revised by (1) using a FLAG-affinity column to pull down Hcf106 variants, including crosslinked OE17-T115C which would be tritiated and visible with fluorography and SDS-PAGE. Any un-crosslinked Hcf106 would not be visible due to its lack of a radiolabel. (2) The SDS-PAGE sample buffer in the original protocol could also be altered by removing the 2- Mercaptoethanol (BME) so the disulfide crosslinks would not be reduced and crosslinked OE17- T115C would appear at a higher molecular weight than un-crosslinked OE17-T115C.

From this study, Hcf106 may have a role handing off the precursor to Tha4 for transport in addition to recognizing the precursor in the receptor complex. From biophysical studies it is also plausible that Hcf106, along with Tha4, destabilize the membrane for transport through substrate-induced conformational changes, which would support the “membrane weakening” model instead of the “pore” model for transport. In a simulation, oligomers of E. coli TatA destabilized lipid bilayers, and an NMR structural analysis showed that the TMD compresses the membrane, after a conformational change that pulls the TMD toward the cytoplasm (Rodriguez et al. 2013), causing hydrophobic mismatch and weakening the membrane. In vivo, fluorescence quenching data linked conformational changes involving the N-terminus and APH region of TatA to proton leakage (membrane weakening) from the membrane. Substrate-induced conformational changes in Hcf106, the homologue of Tha4, are also suspected to cause membrane destabilization. Assays using cysteine accessibility labeling revealed that the N-proximal APH of Hcf106 becomes less accessible to the stroma, angling into the membrane upon substrate-binding in becoming less 54

accessible to the stroma in Habtemichael et al. (2017) The same technique was used in Zhang et al. (2015) where the N-proximal end of the APH became more exposed to the stroma and more parallel with the membrane upon substrate-binding, which was similar to the conformational changes for Tha4 in Hou et al. (2018) where the TMD was pulled towards the cytoplasm. The N- proximal APH region of Hcf106 may prevent hydrophobic mismatch between the short TMD and the lipid bilayer when Hcf106 is not bound to the precursor. Upon precursor binding, the pull of the N-proximal APH region away from the membrane could compress the membrane to match the short hydrophobic region of the TMD. The combined conformational changes of Hcf106 and Tha4 oligomers could compress the bilayer enough to rupture the membrane and transport the precursor.

2.8 REFERENCES

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