BackgroundWhile cellular membranes serve to compartmentalize biochemical reactions to specific microenvironments, the communication and exchange of molecules between the various compartments is essential for cell vitality. The transport of large molecules such as proteins, nucleic acids and ribonucleoprotein complexes requires elaborate molecular machines to prevent compromising the membranes' role as a permeability barrier to ions, metabolic intermediates, and other macromolecules. We seek to explore how these permeability barriers are maintained during the transport of large macromolecules by dissecting the detailed molecular function of three different transport machineries:
The Nuclear Pore Complex
The nuclear pore complex (NPC) spanning the double-membrane nuclear envelope fulfills the essential physiological role of facilitating and regulating the extensive trafficking between the cytoplasm and nucleoplasm in eukaryotic cells. It is unique among transporters because it regulates trafficking of diverse substrates in both directions and it can accommodate passage of molecules as large as ribosomal subunits. Ribosomal subunits, mRNAs, and tRNAs are exported to the cytoplasm. Proteins imported into the nucleus include histones, DNA and RNA polymerases, RNA processing proteins, ribosomal proteins, and numerous transcription factors.
Bacterial Export: The Sec and Tat Machineries
Escherichia coli contain two very different general protein translocation machineries within the cytoplasmic membrane responsible for secretion from the cytoplasm to the periplasm. The Sec machinery (general secretory pathway) translocates polypeptides in an unfolded, linear configuration energetically driven by ATP hydrolysis and the proton motive force, or pmf (consisting of pH and electric field gradients). In contrast, the Tat machinery recognizes and translocates fully-assembled multi-subunit redox proteins with energetic input from the pmf alone. While the Sec machinery is an efficient protein translocator, it is not able to accommodate proteins that must be folded and assembled in the cytoplasm; the Tat machinery fulfills this role instead. The presence of these two very different protein translocation systems in the same biological membrane is a significant advantage to our experimental strategy. Since our transport assays utilize native E. coli membranes, changing the substrate and a few reagents allows examination of the other pathway and expertise can be easily shared between projects.
We seek answers to such basic questions as:
- How long does the transport process take, and what determines translocation rate?
- What intermediate transport steps can be identified?
- How does cargo size, shape, and/or conformational state affect recognition and transport?
- What are the molecular interactions between the cargo and the transport machinery required for transport?
- How is the leakage of ions and molecules minimized?
- And ultimately, what do the answers to all of the above questions imply about the mechanism(s) of transport?
Our goal is to develop detailed, molecular-scale, kinetic models of the above protein transport processes. My laboratory is predominantly a biophysics lab, that is, we seek to understand biological reaction mechanisms by characterizing their physical properties on a molecular level. While we utilize standard biochemical and molecular biological techniques to construct, overexpress, purify and characterize various proteins, mutants and their interactions, our primary investigative tool is fluorescence. We currently use steady-state fluorescence and single molecule narrow-field epifluorescence microscopy, a technique that we have developed (Pub18). We are currently developing and applying fluorescence-based single turnover and single molecule assays that (will) provide millisecond- and nanometer-scale resolution for the above three protein transport systems. Recent accomplishments include:
- We have developed single molecule fluorescence (SMF) and nanometer-scale tracking techniques to elucidate NPC trafficking dynamics in vitro and in vivo with 2 ms and ~40 nm resolution (Pub16, Pub18, and Pub19). We have further expanded these approaches to monitor protein complex assembly and disassembly using single molecule fluorescence resonance energy transfer (smFRET) (Pub22).
- We have demonstrated that the nuclear transport time and transport efficiency are strongly dependent on conditions, and have postulated that these parameters can be regulated in response to cellular transport needs (Pub19).
- We have determined that the intrinsic curvature of lipids plays a critical role in the ability of vesicles to rupture and fuse to form a planar bilayer on a glass surface (Pub17 and Pub20). These results provide an essential set of groundwork experiments for developing single molecule planar bilayer approaches to study the Sec and Tat bacterial transport systems.
- We have developed an in vitro transport assay for the bacterial Tat machinery (Pub21), and demonstrated that a lipid-bound form of the Tat precursor pre-SufI is a functional intermediate in the transport cycle (Pub24).
- We have developed a real-time fluorescence-based transport assay for the bacterial Sec machinery. Using this assay, the observed transport kinetics do NOT support a mechanism in which the precursor protein is methodically transported in discrete steps driven by cycles of SecA conformational changes. Instead, we suggest that precursor movement through the SecYEG channel is primarily driven by Brownian motion (Pub23).