Proteasomes tether to two distinct sites at the nuclear pore complex

Abstract

The partitioning of cellular components between the nucleus and cytoplasm is the defining feature of eukaryotic life. The nuclear pore complex (NPC) selectively gates the transport of macromolecules between these compartments, but it is unknown whether surveillance mechanisms exist to reinforce this function. By leveraging in situ cryo-electron tomography to image the native cellular environment of Chlamydomonas reinhardtii, we observed that nuclear 26S proteasomes crowd around NPCs. Through a combination of subtomogram averaging and nanometer-precision localization, we identified two classes of proteasomes tethered via their Rpn9 subunits to two specific NPC locations: binding sites on the NPC basket that reflect its eightfold symmetry and more abundant binding sites at the inner nuclear membrane that encircle the NPC. These basket-tethered and membrane-tethered proteasomes, which have similar substrate-processing state frequencies as proteasomes elsewhere in the cell, are ideally positioned to regulate transcription and perform quality control of both soluble and membrane proteins transiting the NPC.

Keywords: cryo-electron tomography; focused ion beam; nuclear pore complex; proteasome; quality control.

Fig. 1.

Nuclear proteasomes cluster at the INM in Chlamydomonas. (A) Overview slice through a tomogram spanning the nucleus and cytoplasm (yellow arrows indicate proteasomes; red arrows indicate NPCs) and (B) corresponding segmentation, containing subtomogram averages of proteasomes (blue), NPCs (purple), ATP synthases (yellow), and ribosomes (white, 40S; dark gray, 60S), along with membranes of the nuclear envelope/ER (gray) and mitochondria (orange). (C–E) Close-up views of nuclear proteasomes at the INM: (C) double-capped side views, (D) single-capped side view, and (E) top views. (F) Proteasome concentration in concentric shells expanding from the two membranes of the nuclear envelope (zero point) into the cytoplasm (Left) and nucleus (Right). Values are summed from 61 tomograms. (Inset) Enlarged plot of the two distinct peaks of concentration near the INM. (Scale bars: 200 nm in A, 50 nm in C–E.)

Fig. 2.

Structural classification of the proteasome population. Subtomogram averages of (A) 26S assembly states: double-capped (purple, 21.9-Å resolution) and single-capped (lavender, 23.8-Å resolution); (B) 19S functional states: ground state (green, 17.2-Å resolution) and substrate-processing state (pink, 19.6-Å resolution); (C) 19S binding states: free (blue, 23.8-Å resolution), basket-tethered (yellow, 34.5-Å resolution), and membrane-tethered (orange, 24.9-Å resolution). In B and C, proteasomes were cut in half, and each cap was classified individually. (D) Population statistics of proteasome states within the nucleus (Upper row) and the cytoplasm (Lower row). “?” are unknown functional states that were not clearly classified. (E and F) Focused alignment of the extra densities bound to the proteasome in the (E) basket-tethered and (F) membrane-tethered states. (Insets) Refined tether structures (see also Fig. S4). Fitting a molecular model of the ground state proteasome (26) into the EM density maps shows that both extra densities bind at Rpn9 (light green).

Fig. 3.

Proteasomes bind two distinct sites at NPCs. (A) Proteasome Rpn9 positions relative to the NPC center and INM. There is no positional overlap between basket-tethered and membrane-tethered classes. All proteasomes in the dataset were plotted. Uncertainty in estimating the NPC center position for incomplete NPCs resulted in dispersion of proteasome positions along the x axis. NR, nuclear ring; ONM, outer nuclear membrane. (B) Heatmaps for each binding state, showing proteasome Rpn9 positions symmetrized around the NPC (black silhouette). Orthographic views facing the NPC from the nucleus. Heatmaps were normalized by the number of proteasomes in each class. Only proteasomes adjacent to NPCs with >75% of their structures contained within the tomogram were plotted, resulting in more precise localization. Double-capped proteasomes were plotted only once, using the tethered cap if applicable. (Scale bar: 50 nm.) (C) Close-up orthographic views of structures mapped into three different tomograms, showing NPCs (purple) surrounded by free (blue), basket-tethered (yellow), and membrane-tethered (orange) proteasomes. (Top row) Views from the nucleus; (Bottom row) views along the nuclear envelope (gray).

Fig. 4.

Proteasomes tether to the NPC nuclear basket. (A–H) Sequential slices through a tomographic volume of an NPC (from Fig. 1A, rotated 180°). Thin filaments (red arrows) extend from the NPC into the nucleoplasm. Some filaments are straight, while others are slightly curved. The filament in E appears to be buckled. Attachment of the filaments to basket-tethered proteasomes is visible in slices B, D, and F. In C and G, the attached proteasome is out of the plane of the slice. (Scale bar: 50 nm.) (I) Segmentation of the filaments (red) from A–H shows that they connect the basket-tethered proteasomes (yellow) to the NPC (purple). Perspective view from the nucleus. (J) NPCs (white) mapped into tomograms along with hybrid structures of basket-tethered proteasomes (yellow) fused with the focused alignment of this class’s extra density (green and blue). The extra densities align in a round shape. Orthographic views from the nucleus. (K) For both examples in J, the positions of tethered Rpn9 (X) were fit with an ellipse, reconstituting the shape of the flexible NPC basket. Asterisks indicate ellipse geometrical centers.

Fig. 5.

Proposed quality control functions for proteasomes at the NPC. Basket-tethered and membrane-tethered proteasomes may perform surveillance of transiting soluble and membrane proteins, respectively (blue). The abundant NPC-tethered proteasomes could also serve as a degradation center where nuclear and cytoplasmic proteins are sent for destruction (pink). This degradation center may also function in transcriptional control.