Nucleoplasmic signals promote directed transmembrane protein import simultaneously via multiple channels of nuclear pores

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.

Small heat-shock protein HSPB3 promotes myogenesis by regulating the lamin B receptor

“…LBR is synthesized in the endoplasmic reticulum (ER) and transits through the nuclear pore complex (NPC) to the INM. The first transmembrane region of LBR is sufficient for sorting to the INM and its N-terminus contains an NLS and an intrinsically disordered domain that regulate transit through the NPC 28 – 30 .…”

“…LBR is synthesized in the ER surface and reaches the INM passing through the nuclear pores 30 , 38 . Within the INM, LBR molecules oligomerize and organize in multiple microdomains that contain immobile oligomerized LBR, while nonoligomerized LBR is mostly mobile 34 , 62 .…”

STING Nuclear Partners Contribute to Innate Immune Signalling Responses

“…That the INM STING pool can mobilize to translocate to the ONM through the peripheral channels of the NPC may reflect a backup mechanism to signal IIR responses using the peripheral NPC channels when viruses inhibit central channel transport. Viruses often target the central channel of the NPCs to either block transport or to usurp it so that virus transcripts are preferentially transported over host-directed transport 71,72,73,74 , but the peripheral channels are normally used for membrane protein transport 38,42,43,75 so that STING as a multi-spanning transmembrane protein could bypass this block to signal IIR. This does not preclude the well-established STING signaling cascades from the ER/Golgi compartment normally using the central channel of the NPC -indeed IRF3 is known to translocate through the NPC central channel 76,77 , but our findings of increased STING mobility and nucleo-cytoplasmic shuttling during IIR through the peripheral NPC channels suggest that STING may provide a backup system for activating IIR when central channel transport is disrupted.…”

“…We next turned to a different super resolution approach, Single-Molecule Fluorescence Recovery After Photobleaching (smFRAP) microscopy, that enables the tracking of individual NETs as they diffuse along the INM and ONM of the NE 42,43,44 . The nuclear pool of STING-GFP redistributed out of the nucleus upon stimulation of the cells with poly(I:C) or dsDNA ( Fig.…”

Protocol for single-molecule fluorescence recovery after photobleaching microscopy to analyze the dynamics and spatial locations of nuclear transmembrane proteins in live cells

“…For complete details on the use and execution of this protocol, please refer to Mudumbi et al. (2016a , 2016b , 2020 .…”