Direct Interaction between a Precursor Mature Domain and Transport Component Tha4 during Twin Arginine Transport of Chloroplasts    

Abstract

Proteins destined for the thylakoid lumen of chloroplasts must cross three membranes en route. The chloroplast twin arginine translocation (cpTat) system facilitates the transport of about one-half of all proteins that cross the thylakoid membrane in chloroplasts. Known mechanistic features of the cpTat system are drastically different from other known translocation systems, notably in its formation of a transient complex to transport fully folded proteins utilizing only the protonmotive force generated during photosynthesis for energy. However, key details, such as the structure and composition of the translocation pore, are still unknown. One of the three transmembrane cpTat components, Tha4, is thought to function as the pore by forming an oligomer. Yet, little is known about the topology of Tha4 in thylakoid, and little work has been done to detect precursor-Tha4 interactions, which are expected if Tha4 is the pore. Here, we present evidence of the interaction of the precursor with Tha4 under conditions leading to transport, using cysteine substitutions on the precursor and Tha4 and disulfide bond formation in pea (Pisum sativum). The mature domain of a transport-competent precursor interacts with the amphipathic helix and amino terminus of functional Tha4 under conditions leading to transport. Detergent solubilization of thylakoids post cross linking and blue-native polyacrylamide gel electrophoresis analysis shows that Tha4 is found in a complex containing precursor and Hcf106 (i.e. the cpTat translocase). Affinity precipitation of the cross-linked complex via Tha4 clearly demonstrates that the interaction is with full-length precursor. How these data suggest a role for Tha4 in cpTat transport is discussed.

Structural model for the protein-translocating element of the twin-arginine transport system

“…The TatA oligomer structure suggests that the APHs form a platform upon which the substrate lies, an arrangement that is consistent with cross-linking studies (17,47,48). Nonspecific substrate-APH interactions would then provide cross-bridging contacts between APHs that could assist in stabilizing the TatA oligomer.…”

The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding

“…As the switch of the APH conformation is a consequence of a substrate interaction, the APH itself is the most likely interaction site. An APH interaction with mature domains of Tat substrates had been for the first time experimentally demonstrated by the group of Carole Dabney Smith for the thylakoidal system (46). Also in the E. coli system, mature domains of Tat substrates have been shown to interact with TatA (16,(47)(48)(49), and it has been demonstrated that TatA has the capacity to interact with Tat substrates in a TatBC independent manner (26).…”

“…Also in the E. coli system, mature domains of Tat substrates have been shown to interact with TatA (16,(47)(48)(49), and it has been demonstrated that TatA has the capacity to interact with Tat substrates in a TatBC independent manner (26). For the thylakoid system it has been suggested that the APH interactions relate to nonspecific passive contacts before or during the membrane passage of mature domains, as a crosslink to a position at the end of the APH (Phe-48) was TatB-and PMF-dependent (46). In agreement with the thylakoidal system data, we also found no substrate effects at the C-terminal end of the APH in the absence of TatBC ( Figure 6F).…”

“…This may be (i) TatBC itself -which changes conformation upon substrate binding -or it may be (ii) the substrate, which might tightly associate to this region of the TatA-APH. Both scenarios are conceivable and a direct substrate interaction with the APH has been already demonstrated (46). Also the substrate induced accessibility differences that have been detected in the thylakoid system were considered to potentially indicate a steric hindrance by substrate (33).…”

“…Based on the substrateindependent TatA/TatBC interactions, we currently favor the view that TatA and TatBC always function hand in hand and remain in close proximity. The RR-motif of Tat signal peptides can remain associated with TatC during translocation (15), and Tat substrates are contacting TatA during transport with their mature domains (46,49,26). TatA recognizes the transported domains to initiate the conformational switch, but TatBC recognizes the RR-motif and transport is only achieved when both activities cooperate.…”