Abstract
Roughly 10% of eukaryotic transmembrane proteins are found on the nuclear membrane, yet how such proteins target and translocate to the nucleus remains in dispute. Most models propose transport through the nuclear pore complexes, but a central outstanding question is whether transit occurs through their central or peripheral channels. Using live-cell high-speed super-resolution single-molecule microscopy we could distinguish protein translocation through the central and peripheral channels, finding that most inner nuclear membrane proteins use only the peripheral channels, but some apparently extend intrinsically disordered domains containing nuclear localization signals into the central channel for directed nuclear transport. These nucleoplasmic signals are critical for central channel transport as their mutation blocks use of the central channels; however, the mutated proteins can still complete their translocation using only the peripheral channels, albeit at a reduced rate. Such proteins can still translocate using only the peripheral channels when central channel is blocked, but blocking the peripheral channels blocks translocation through both channels. This suggests that peripheral channel transport is the default mechanism that was adapted in evolution to include aspects of receptor-mediated central channel transport for directed trafficking of certain membrane proteins.
Highlights
10% of eukaryotic transmembrane proteins are found on the nuclear membrane, yet how such proteins target and translocate to the nucleus remains in dispute
To test the two nuclear pore complexes (NPCs)-dependent transport route models (Fig. 1a and Supplementary Fig. 1), we selected several endogenous nuclear envelope transmembrane (NET) with a range of characteristics to determine if different NETs use distinct transport routes, and if so, what characteristics direct the use of a particular route
By using in vivo imaging, we showed the existence of functional peripheral channels in the NPC, and how they are used by INM proteins to diffuse from the ONM into the INM
Summary
10% of eukaryotic transmembrane proteins are found on the nuclear membrane, yet how such proteins target and translocate to the nucleus remains in dispute. For NPC-dependent transport, this suggests that transit might occur through ~10 nm wide peripheral channels of the NPC that were identified by early electron microscopy (EM) studies roughly 30 years ago[10,25,26]; the exact location, dimensions, and functionality of these channels has remained unclear[27,28,29] Functional confirmation of this peripheral channel transport route is of wide interest because many pathogens are known to disrupt central channel transport[30,31,32,33,34,35,36], and in such cases the peripheral channels could function as a critical backup mechanism for nuclear signaling. This study assesses two NPC-dependent models: free lateral diffusion-retention and nuclear localization signal (NLS)-dependent facilitated transport Both models require that the transmembrane domain of INM proteins stays embedded in the nuclear envelope during transit from the ONM to the INM, but there are some critical differences between the two mechanisms. SPEED microscopy combines high-speed 2D single-molecule localization microscopy and a post-localization 2D to 3D transformation algorithm, in which the former records single-molecule locations of transmembrane proteins through the NPCs with a spatiotemporal resolution of
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