Role of Molecular Charge in Nucleocytoplasmic Transport

Abstract

Transport of genetic materials and proteins between the nucleus and cytoplasm of eukaryotic cells is mediated by nuclear pore complexes (NPCs). A selective barrier formed by phenylalanine-glycine (FG) nucleoporins (Nups) with net positive charges in the NPC allows for passive diffusion of signal-independent small molecules and transport-receptor facilitated translocation of signal-dependent cargo molecules. Recently, negative surface charge was postulated to be another essential criterion for selective passage through the NPC. However, the charge-driven mechanism in determining the transport kinetics and spatial transport route for either passive diffusion or facilitated translocation remains obscure. Here we employed high-speed single-molecule fluorescence microscopy with an unprecedented spatiotemporal resolution of 9 nm and 400 µs to uncover these mechanistic fundamentals for nuclear transport of charged substrates through native NPCs. We found that electrostatic interaction between negative surface charges on transiting molecules and the positively charged FG Nups, although enhancing their probability of binding to the NPC, never plays a dominant role in determining their nuclear transport mode or spatial transport route. A 3D reconstruction of transport routes revealed that small signal-dependent endogenous cargo protein constructs with high positive surface charges that are destined to the nucleus, rather than repelled from the NPC as suggested in previous models, passively diffused through an axial central channel of the NPC in the absence of transport receptors. Finally, we postulated a comprehensive map of interactions between transiting molecules and FG Nups during nucleocytoplasmic transport by combining the effects of molecular size, signal and surface charge.

Deciphering the Structure and Function of Nuclear Pores Using Single-Molecule Fluorescence Approaches

“…For example, the simulations in Figure 10 illustrate the role of measurement error in SPEED analysis. Importantly, however, comparison of SPEED generated particle distribution maps for signal-independent and -dependent particles (the raw 2D data rather than computer-generated 3D maps) reveals clear differences in widths at the NPC central pore, 79,110,120,186 implying that the two transport pathways use different parts of the FG-network. That is, signal-independent cargos access a significantly smaller region within the central pore, consistent with the central channel hypothesis.…”

Particle Transport through Hydrogels Is Charge Asymmetric

“…To reach specific loci in target cells, molecules of interest must traverse complex surroundings consisting of a crowded, interacting environment of biomacromolecules. Molecule diffusion through biological gels such as the cytoplasm, mucus, nuclear pore complex, or the extracellular matrix (ECM) is dictated by the local environment and critical for proper functioning of cell processes (1)(2)(3)(4)(5). Due to their importance as protective barriers against viruses, bacteria, and toxic agents, there has been substantial research in recent years to obtain a better understanding of the transport processes governing the diffusion and penetration of particles through biogels.…”

Asthma and allergic rhinitis associate with the rs2229542 variant that induces a p.Lys90Glu mutation and compromises AKR1B1 protein levels

“…According to the available crystal structure (1PWL), p.Lys90 is surface‐exposed, and hence, putatively involved in interactions or subjected to posttranslational modifications. The organization of charged, polar, and polarizable residues in the tertiary structure of a protein creates large internal electrostatic fields, expected to affect many aspects of protein function, including molecular interactions and subcellular localization (Goryaynov & Yang, ; Reytor et al., ; Valero et al., ). The available AKR1B1 structure (Figure E and F) shows the amino group at p.Lys90 lateral chain surrounded by negative charges.…”