Molecular Mechanism of Protein Transport to Biological Membranes
Proper localization of proteins is essential to establish and maintain order and organization in all cells. The signal recognition particle (SRP) and its receptor (SR) constitute the major cellular machinery that delivers nascent proteins to the eukaryotic ER membrane, or the bacterial plasma membrane. Our goal is to decipher, at a biochemical and biophysical level, the intricate inner workings of this universally conserved protein transport machinery, with a particular focus on understanding how two closely related and directly interacting GTPases in the SRP and SR use their cycles of GTP binding and hydrolysis to promote the efficiency and fidelity of protein transport. Our previous work showed that, unlike classical signaling GTPases that undergo a simple two-state switch between GTP- and GDP-bound conformations, SRP and SR undergo a series of discrete conformational changes when they interact with each other, culminating in the reciprocal activation of GTP hydrolysis on both GTPases. We now propose to delineate how these corformational changes are used to drive and regulate the protein targeting reaction. To this end, we will define whether, when and how the cargo protein and target membrane affect the GTPase cycles of SRP and SR and their conformational changes. We will also determine how the GTPase cycles of SRP and SR modulate the individual steps - cargo recognition, delivery to the membrane, and cargo transfer to the translocation channel - during transport of the cargo protein to the target membrane. A likely link between the GTPase cycles and protein transport is the SRP RNA: it facilitates SRP-SR association, and the binding sites for cargo protein and for the SRP RNA are located in close proximity in the same domain of SRP. To elucidate the role of the SRP RNA, we will isolate mutant RNAs that block protein transport at various stages using a combination of in vitro selections and in vivo screens. We will then determine whether the mutant RNAs are defective in catalyzing the SRP-SR interaction, in mediating the SRP-cargo interactions, or in the coupling between these two functions. Insights into the role of the SRP GTPases and the SRP RNA can also be gained from comparison with a novel chloroplast (cp) SRP system. This system uses a protein, cpSRP43, in place of the SRP RNA, and employs a posttranslational mode of targeting in place of the co-translational mode. We will examine the role of SRP RNA and I cpSRP43 in their respective pathways and search for conditions that allow them to cross-complement. We will also compare the interaction of cpSRP with its cargo protein, which lacks a translating ribosome, with that of canonical SRPs that recognize the ribosome together with newly synthesized proteins. Ultimately, these studies will not only address the basic question of protein localization within the cell, but also new insights into the general principles of molecular recognition and regulation.
Arnold O. Beckman exemplifies the meaning of the word humanitarian. Combined with his unwavering enthusiasm for life, his keen sense of humor and his strong moral and ethical principles, he is a national icon.