Siegfried M. Musser

Nuclear import time and transport efficiency depend on importin β concentration

Although many components and reaction steps necessary for bidirectional transport across the nuclear envelope (NE) have been characterized, the mechanism and control of cargo migration through nuclear pore complexes (NPCs) remain poorly understood. Single-molecule fluorescence microscopy was used to track the movement of cargos before, during, and after their interactions with NPCs. At low importin β concentrations, about half of the signal-dependent cargos that interacted with an NPC were translocated across the NE, indicating a nuclear import efficiency of ∼50%. At high importin β concentrations, the import efficiency increased to ∼80% and the transit speed increased approximately sevenfold. The transit speed and import efficiency of a signal-independent cargo was also increased by high importin β concentrations. These results demonstrate that maximum nucleocytoplasmic transport velocities can be modulated by at least ∼10-fold by the importin β concentration and therefore suggest a potential mechanism for regulating the speed of cargo traffic across the NE.

Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.

The twin-arginine translocation (Tat) pathway in Escherichia coli transports fully folded and assembled proteins across the energy-transducing periplasmic membrane. In chloroplasts, Tat transport requires energy input only from the proton motive force. To elucidate the mechanism and energetics of bacterial Tat protein transport, we developed an efficient in vitro transport assay using TatABC-enriched inverted membrane vesicles and the physiological precursor pre-SufI. We report transport efficiencies of 60–80% for nanomolar pre-SufI concentrations. Dissipation of the pH gradient does not reduce pre-SufI transport efficiency. Instead, pre-SufI transport requires at least two electrical potential (Δψ)–dependent steps that differ in both the duration and minimum magnitude of the required Δψ. The data are consistent with a model in which a substantial Δψ of short duration is required for an early transport step, and in which a small Δψ of long duration is necessary to drive a later transport step.

Interconvertibility of Lipid- and Translocon-bound Forms of the Bacterial Tat Precursor pre-SufI

Signal peptides target protein cargos for secretion from the bacterial cytoplasm. These signal peptides contain a tri‐partite structure consisting of a central hydrophobic domain (h‐domain), and two flanking polar domains. Using a recently developed in vitro transport assay, we report here that a central h‐domain position (C17) of the twin arginine translocation (Tat) substrate pre‐SufI is especially sensitive to amino acid hydrophobicity. The C17I mutant is transported more efficiently than wild type, whereas charged substitutions completely block transport. Transport efficiency is well‐correlated with Tat translocon binding efficiency. The precursor protein also binds to non‐Tat components of the membrane, presumably to the lipids. This lipid‐bound precursor can be chased through the Tat translocons under conditions of high proton motive force. Thus, the non‐Tat bound form of the precursor is a functional intermediate in the transport cycle. This intermediate appears to directly equilibrate with the translocon‐bound form of the precursor.

Single-molecule measurements of importin α/cargo complex dissociation at the nuclear pore

Macromolecules are transported between the cytoplasm and the nucleoplasm of eukaryotic cells through nuclear pore complexes (NPCs). Large (more than ≈40 kDa) transport cargoes imported into the nucleus typically form a complex with at least one soluble transport cofactor of the importin (Imp) β superfamily. Many cargoes require an accessory cofactor, Imp α, which binds to Imp β and to the nuclear localization sequence on the cargo. We previously reported the use of narrow-field epifluorescence microscopy to directly monitor cargoes in transit through NPCs in permeabilized cells. We now report an expanded approach in which single-molecule fluorescence resonance energy transfer (FRET) is used to detect the disassembly of Imp α/cargo complexes as they transit through NPCs. We found that CAS, the recycling cofactor for Imp α, and RanGTP are essential for this dissociation process. After Imp α/cargo complex dissociation, most Imp α and cargo molecules entered the nucleoplasm. In contrast, the majority of Imp α/cargo complexes that did not dissociate at the NPC in the presence of CAS and RanGTP returned to the cytoplasm. These data are consistent with a model in which Imp α/cargo complexes are dissociated on the nucleoplasmic side of the NPC, and this dissociation requires both CAS and RanGTP.

Large cargo transport by nuclear pores: implications for the spatial organization of FG‐nucleoporins

Nuclear pore complexes (NPCs) mediate cargo traffic between the nucleus and the cytoplasm of eukaryotic cells. Nuclear transport receptors (NTRs) carry cargos through NPCs by transiently binding to phenylalanine‐glycine (FG) repeats on intrinsically disordered polypeptides decorating the NPCs. Major impediments to understand the transport mechanism are the thousands of FG binding sites on each NPC, whose spatial distribution is unknown, and multiple binding sites per NTR, which leads to multivalent interactions. Using single molecule fluorescence microscopy, we show that multiple NTR molecules are required for efficient transport of a large cargo, while a single NTR promotes binding to the NPC but not transport. Particle trajectories and theoretical modelling reveal a crucial role for multivalent NTR interactions with the FG network and indicate a non‐uniform FG repeat distribution. A quantitative model is developed wherein the cytoplasmic side of the pore is characterized by a low effective concentration of free FG repeats and a weak FG‐NTR affinity, and the centrally located dense permeability barrier is overcome by multivalent interactions, which provide the affinity necessary to permeate the barrier.

Single molecule studies of nucleocytoplasmic transport.

Molecular traffic between the cytoplasm and the nucleoplasm of eukaryotic cells is mediated by nuclear pore complexes (NPCs). Hundreds, if not thousands, of molecules interact with and transit through each NPC every second. The pore is blocked by a permeability barrier, which consists of a network of intrinsically unfolded polypeptides containing thousands of phenylalanine-glycine (FG) repeat motifs. This FG-network rejects larger molecules and admits smaller molecules or cargos bound to nuclear transport receptors (NTRs). For a cargo transport complex, minimally consisting of a cargo molecule plus an NTR, access to the permeability barrier is provided by interactions between the NTR and the FG repeat motifs. Numerous models have been postulated to explain the controlled accessibility and the transport characteristics of the FG-network, but the amorphous, flexible nature of this structure has hindered characterization. A relatively recent development is the ability to monitor the real-time movement of single molecules through individual NPCs via single molecule fluorescence (SMF) microscopy. A major advantage of this approach is that it can be used to continuously monitor a series of specific molecular interactions in an active pore with millisecond time resolution, which therefore allows one to distinguish between kinetic and thermodynamic control. Novel insights and prospects for the future are outlined in this review. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.

Comparison of ubiquinol and cytochrome c terminal oxidases An alternative view

There have been numerous instances in the recent literature where the properties of ubiquinol and cytochrome c terminal oxidases are compared. Here we specifically examine the cytochrome bo 3‐type ubiquinol oxidase from Escherichia coli and the cytochrome aa 3‐type cytochrome c oxidases. A second redox‐active copper site (CuA) is present only in the cytochrome c oxidases and the physiological electron donors for the two enzymes are different (ubiquinol‐8 vs. ferrocytochrome c). In our opinion, these differences are significant and most likely indicate that distinct turnover mechanisms are operative in the two enzymes.

Kinetics of Precursor Interactions with the Bacterial Tat Translocase Detected by Real-time FRET

Background: The Tat machinery transports folded proteins from the bacterial cytoplasm to the periplasm. Results: A Δψ is required for a Tat cargo to move away from the TatBC receptor complex. Conclusion: Cargo migration away from the TatBC complex requires a Δψ-dependent assembly step or conformational change. Significance: Cargo migration from the TatBC receptor is a major rate-limiting step of Tat transport. The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. Traditionally, in vitro protein translocation studies have been performed using gel-based transport assays. This technique suffers from low time resolution, and often, an inability to distinguish between different steps in a continuously occurring translocation process. To address these limitations, we have developed an in vitro FRET-based assay that reports on an early step in the Tat translocation process in real-time. The natural Tat substrate pre-SufI was labeled with Alexa532 (donor), and the fluorescent protein mCherry (acceptor) was fused to the C terminus of TatB or TatC. The colored Tat proteins were easily visible during purification, enabling identification of a highly active inverted membrane vesicle (IMV) fraction yielding transport rates with NADH almost an order of magnitude faster than previously reported. When pre-SufI was bound to the translocon, FRET was observed for both Tat proteins. FRET was diminished upon addition of nonfluorescent pre-SufI, indicating that the initial binding step is reversible. When the membranes were energized with NADH, the FRET signal was lost after a short delay. These data suggest a model in which a Tat cargo initially associates with the TatBC complex, and an electric field gradient is required for the cargo to proceed to the next stage of transport. This cargo migration away from the TatBC complex requires a significant fraction of the total transport time.

Evolution of the Cytochrome c Oxidase Proton Pump

Abstract. The superfamily of quinol and cytochrome c terminal oxidase complexes is related by a homologous subunit containing six positionally conserved histidines that ligate a low-spin heme and a heme–copper dioxygen activating and reduction center. On the basis of the structural similarities of these enzymes, it has been postulated that all members of this superfamily catalyze proton translocation by similar mechanisms and that the CuA center found in most cytochrome c oxidase complexes serves merely as an electron conduit shuttling electrons from ferrocytochrome c into the hydrophobic core of the enzyme. The recent characterization of cytochrome c oxidase complexes and structurally similar cytochrome c:nitric oxide oxidoreductase complexes without CuA centers has strengthened this view. However, recent experimental evidence has shown that there are two ubiquinone(ol) binding sites on the Escherichia coli cytochrome bo3 complex in dynamic equilibrium with the ubiquinone(ol) pool, thereby strengthening the argument for a Q(H2)-loop mechanism of proton translocation [Musser SM et al. (1997) Biochemistry 36:894–902]. In addition, a number of reports suggest that a Q(H2)-loop or another alternate proton translocation mechanism distinct from the mitochondrial aa3-type proton pump functions in Sulfolobus acidocaldarius terminal oxidase complexes. The possibility that a primitive quinol oxidase complex evolved to yield two separate complexes, the cytochrome bc1 and cytochrome c oxidase complexes, is explored here. This idea is the basis for an evolutionary tree constructed using the notion that respiratory complexity and efficiency progressively increased throughout the evolutionary process. The analysis suggests that oxygenic respiration is quite an old process and, in fact, predates nitrogenic respiration as well as reaction-center photosynthesis.

Bacterial Sec Protein Transport Is Rate-limited by Precursor Length: A Single Turnover Study

An in vitro real-time single turnover assay for the Escherichia coli Sec transport system was developed based on fluorescence dequenching. This assay corrects for the fluorescence quenching that occurs when fluorescent precursor proteins are transported into the lumen of inverted membrane vesicles. We found that 1) the kinetics were well fit by a single exponential, even when the ATP concentration was rate-limiting; 2) ATP hydrolysis occurred during most of the observable reaction period; and 3) longer precursor proteins transported more slowly than shorter precursor proteins. If protein transport through the SecYEG pore is the rate-limiting step of transport, which seems likely, these conclusions argue against a model in which precursor movement through the SecYEG translocon is mechanically driven by a series of rate-limiting, discrete translocation steps that result from conformational cycling of the SecA ATPase. Instead, we propose that precursor movement results predominantly from Brownian motion and that the SecA ATPase regulates pore accessibility.