Research

In the post-genomic era, there has been tremendous progress in the identification of genes and pathways for many important cellular processes. However, our understanding of the energetic principles and molecular mechanisms that underlie these processes lags behind. Using the SRP pathway as a model system, research in our lab aims at bridging this gap in our understanding of biology through a combination of approaches including biochemistry, biophysics, molecular and cell biology, protein engineering, and mechanistic enzymology.

Figure 1 GTPase cycle of classical (A) vs. SRP and SR GTPases (B). GEF, nucleotide exchange factor; GAP, GTPase activating protein.

The GTPase superfamily of proteins act as molecular switches to regulate numerous cellular processes, including signal transduction, translation, cytoskeletal organization, protein transport, and cell division. Classical work on signaling GTPases such as Ras has established a 'GTPase switch' paradigm to explain their mode of regulation (Fig. 1A): each GTPase switches between an active, GTP-bound state and an inactive, GDP-bound state. Inter-conversion between the two states is intrinsically slow, and is regulated by external factors (Fig. 1A, GEFs and GAPs). This allows GTPases to switch between 'on' and 'off' states in temporal succession in response to cellular signaling cues.

Figure 2 Protein transport by the SRP. 1, SRP recognizes N-terrminal signal sequences on nascent proteins that emerge from the ribosome. 2, cargo is delivered to the membrane via the SRP-SR interaction. 3, SRP unloads its cargo to a translcation channel (translocon) in the membrane. 4, the cargo proteins is integrated into the membrane or delivered to the secretory pathway.

Two homologous GTPases, one in the signal recognition particle (SRP) and one in the SRP receptor (SR), regulate the transport of newly synthesized proteins to membranes (Fig. 2). Roughly a third of cellular proteins are delivered by the SRP, including insulin, the prion protein, and over 90% of T cell receptors. Thus SRP and SR comprise the major cellular machinery that ensures proper sorting and localization of proteins within a cell. Despite the universal conservation of SRP, little is understood about how it works at a molecular level. Our goal is to elucidate how SRP and SR use their GTPase cycles to provide the vectorial driving force and improve the fidelity of protein transport.

Intriguingly, SRP and SR are a notable exception to the 'GTPase switch' paradigm: they do not undergo significant rearrangements depending on which nucleotide is bound (Fig. 1B, steps 1a-1b), nor do they use external regulatory factors to help them switch between 'on' and 'off' states. Instead, SRP and SR form a stable complex when both of them are GTP-bound (Fig. 1B, step 2), and reciprocally activate each other's GTPase activity in the complex (Fig. 1B, step 3). GTP hydrolysis then drives complex disassembly (Fig. 1B, step 4). The number of GTPases with such exceptional design features is growing rapidly, including elongation factor G that drives protein synthesis, and the dynamin family of GTPases that control endocytosis and mitochondria dynamics. How do these GTPases, which do not undergo a classical 'GTPase switch' and have no external regulatory factors, act as molecular switches to regulate complex cellular processes? Elucidation of the mechanism of SRP will not only provide us a deeper understanding of this fundamental cellular process, but also lead to a new regulatory paradigm for other important cellular processes controlled by non-canonical GTPases.

Through in-depth biochemical and biophysical analyses, we showed that during the interaction cycle between SRP and SR (Fig. 1B, steps 2-4), a series of conformational changes occur that culminate in the activation of GTP hydrolysis in both GTPases (Fig. 3; refs). This led to our new working hypothesis: each rearrangement provides a regulatory point that allows SRP and SR to respond to spatial and temporal cues such as cargo- and membrane-binding, thus ensuring an ordered series of cargo loading, delivery and unloading events at the appropriate stages during protein transport (Fig. 3). A combination of approaches are used to test and expand upon this hypothesis.

Figure 3 Multiple conformational changes in SRP and SR regulate protein transport. 1a & 1b, SRP and SR undergo an open-to-closed rearrangement upon binding the cargo and the target membrane, respectively. 2, Formation of a closed SRP•SR complex delivers the cargo to the membrane surface. 3, Another rearrangement drives cargo transfer to the translocon and leads to GTPase activation. 4, GTP hydrolysis drives SRP•SR complex disassembly. 5, premature GTP hydrolysis aborts protein transport.

We have developed fluorescent probes on SRP and SR that monitor each rearrangement during the SRP-SR interaction in real time. Using these tools, we want to define whether, when and how the 'cargo' and the target membrane modulate these conformational changes. Already, we have observed many interesting effects: (i) SRP-SR binding is accelerated over 20-fold by the cargo and 5-fold by the membrane translocon. Thus consistent with our hypothesis, cargo- and membrane-association modulates the conformation of SRP and SR (Fig. 3, step 1a & 1b) so that they can better bind each other, thereby facilitating cargo delivery to the membrane (step 2). (ii) SR weakens the SRP-cargo interaction, and thus helps drive the unloading of cargo from SRP to the translocon (Fig. 3, step 3). (iii) Most intriguingly, the cargo stalls the SRPoSR complex at early conformational stages and delays the rearrangement that leads to activation of GTP hydrolysis in this complex. Such 'pausing' have been observed in DNA replication and transcription, and often serve as a checkpoint for improving fidelity. We will test whether the 'pausing' we observed in the SRPoSR complex can analogously serve as a fidelity checkpoint, allowing transport of the 'wrong' cargo to abort through premature GTP hydrolysis (Fig. 3, step 5). Given the plethora of effects we have observed with the cargo, we are most excited to test whether the membrane translocon can similarly modulate SRP and SR's conformational changes that serve to ensure the efficiency and fidelity of protein transport.

Figure 4Labeling of nascent chain with a fluorescent dye ().

Many mechanistic questions about the SRP pathway couldn't be addressed due to the lack of quantitative assays that allow the many steps in this pathway to be dissected. To address this issue, we are developing a quantitative assay that will allow us to follow the individual steps during protein transport in real time. Using the amber suppressor tRNA technology, we want to generate a fluorescent 'cargo' for SRP. We will also incorporate FRET donor and acceptors for NBD into purified SRP and secY translocon, respectively. Using tri-color FRET, we can then analyze the kinetics of the individual molecular events, including cargo recognition, delivery and unloading, as protein transport progresses. This assay will allow, for the first time, a rigorous mechanistic analysis of this complex cellular process and address many questions in this pathway. For example, we can use our knowledge of the GTPases and the extensive array of mutant proteins to selectively perturb the GTPase cycle on SRP and SR, and determine at which specific step(s) the protein transport reaction is affected by such perturbations. For another example, we can compare the efficiency of the cargo recognition, delivery and unloading steps for SRP-dependent cargo proteins versus mutant cargos in which the signal sequence is systematically varied. The most intriguing question is whether the SRP pathway uses additional fidelity checkpoints beyond the cargo recognition step: Can SRP-SR docking (Fig. 3, step 2) be accelerated much more substantially by the 'right' than the 'wrong' cargos? Is premature GTP hydrolysis used to abort the transport of 'wrong' cargos (Fig. 3, step 5), akin to kinetic proofreading mechanisms during protein synthesis? These analyses will allow us to have a complete picture of where and how fidelity arises during protein transport, and to know whether the energy from GTP hydrolysis is used to improve fidelity.

A central question about the SRP is how information about cargo loading and unloading in the cargo-binding M-domain is communicated to the GTPase domain of SRP, which, in turn, mediate its interaction with SR. These communications are crucial for ensuring an ordered series of cargo binding, delivery, and release events at the proper stages of protein targeting (Fig. 3). How the M-domain of SRP is positioned relative to its NG domain remains controversial. These two domains are connected by a flexible linker that is often unresolved in crystal structures; and different available structures show various domain arrangements. It is likely that such variability is intrinsic to the SRP and allows the M- and NG-domains to rearrange their relative positions in response to cargo binding and release. In our lab, we use various spectroscopic techniques such as electron paramagnetic resonance spectroscopy (SDSL-EPR) to elucidate both the global domain arrangement of SRP and the dynamic feature of the domain-domain interaction.

A likely link between the GTPase cycles and the targeting reaction is the SRP RNA, because (i) we have shown that the SRP RNA plays a catalytic role in the formation of the SRPoSR complex, accelerating both their association and dissociation by 400-fold; and (ii) the signal sequence and RNA binding sites are located in close proximity within the same domain in SRP. Thus, it can be envisioned that the presence of the signal sequence could modulate the activity of 4.5S RNA, turning it into an active regulator that mediates the coupling between the GTPase cycles and the targeting cycle. We take two approaches to understand the role of the SRP RNA: (1) Using a combination of computational simulation, site-directed mutagenesis, and spectroscopic methods, we want to understand how the SRP RNA exerts its unprecedented catalytic effect on the SRP-SR interaction; (2) We want to isolate and characterize mutant RNAs that are locked into specific conformational states, and are thus defective in mediating the protein targeting reaction at specific stages. Several interesting classes of mutant RNAs can be envisioned: (i) mutants that fail to accelerate SRP-SR complex formation; (ii) mutants that fail to mediate the interaction of SRP with the cargo; (iii) mutants that can mediate both SRP-cargo and SRP-SR interactions, but lose the coupling between the two functions. These mutants will shed light on the role of the SRP RNA in the targeting reaction, and on the molecular mechanism by which communication between the cargo binding and GTPase domains is established.

The cpSRP pathway is responsible for delivering light harvesting chlorophyll-binding proteins to the thylakoid membrane. Intriguingly, this system uses a novel protein, cpSRP43, in place of the otherwise universally conserved SRP RNA, and employs a post-translational mode of targeting in place of the co-translational mode. Thus, the cpSRP system offers a unique opportunity to probe the role of SRP RNA (or cpSRP43) in protein transport. The different modes of substrate utilization in the cpSRP pathway can help us understand the coupling between the protein transport reaction and the GTPase cycles of SRP and SR. We can compare the mode of SRP-substrate interaction in the chloroplast system, which lacks a translating ribosome, with the canonical SRP systems that recognize ribosomeonascent chain complexes. Further, the simplicity of the cargo protein the cpSRP system will provide a simpler, more accessible system to develop quantitative in vitro targeting assays. Thus, the cpSRP pathway offers a complementary and conceivably much faster route to the elucidation of the mechanism by which the GTPase cycles of SRP and SR regulate the protein transport reaction.