Nuclear Transport of Yeast Proteasomes

Abstract

Proteasomes are key proteases in regulating protein homeostasis. Their holo-enzymes are composed of 40 different subunits which are arranged in a proteolytic core (CP) flanked by one to two regulatory particles (RP). Proteasomal proteolysis is essential for the degradation of proteins which control time-sensitive processes like cell cycle progression and stress response. In dividing yeast and human cells, proteasomes are primarily nuclear suggesting that proteasomal proteolysis is mainly required in the nucleus during cell proliferation. In yeast, which have a closed mitosis, proteasomes are imported into the nucleus as immature precursors via the classical import pathway. During quiescence, the reversible absence of proliferation induced by nutrient depletion or growth factor deprivation, proteasomes move from the nucleus into the cytoplasm. In the cytoplasm of quiescent yeast, proteasomes are dissociated into CP and RP and stored in membrane-less cytoplasmic foci, named proteasome storage granules (PSGs). With the resumption of growth, PSGs clear and mature proteasomes are transported into the nucleus by Blm10, a conserved 240 kDa protein and proteasome-intrinsic import receptor. How proteasomes are exported from the nucleus into the cytoplasm is unknown.

Keywords: Blm10; importin; karyopherin; nuclear transport; proteasome; proteasome storage granules.

Figure 1

The transport cycles of importins and exportins in yeast. (A) Import of cNLS (red) containing cargos is mediated by importin αβ. (B) Importin α recognizes and binds the cNLS of the cargo as well as importin β. After transport through the NPC (C) the GTP bound GTPase Ran binds to importin β and dissociates the import complex (D). (E) The export of NES (green) containing cargo from the nucleus is mediated by exportin. In the presence of GTP bound Ran, exportin binds Ran and the cargo (F) and transports the complex across the NPC into the cytoplasm (G). Hydrolysis of GTP to GDP is stimulated by Ran binding protein 1 and Ran GTPase activating protein 1 in the cytoplasm. Conformational rearrangements in RanGDP lead to the dissociation of the exportin/RanGDP/cargo complex (H).

Figure 2

True-to-scale representation of proteasomal subcomplexes, NLS conferring adaptor proteins and nuclear transport receptors. The 20S CP, Pba1-2 bound 15S precursor complex, 19S base and 19S lid complexes are depicted with putative cNLS in α1, α4, α5, Rpn2, and Rpt2 colored in red. Importin αβ (Srp1/Kap95) binds to 20S, 15S, and 19S base complexes during nuclear import. Sts1/Cut8 binds to the 19S lid complex and possibly provides a cNLS, depicted in red, for importin αβ dependent nuclear import. Blm10 can bind to 20S and 15S complexes and possibly acts as an import receptor for nuclear transport.

Figure 3

Cellular localization of proteasomal subunits. (A) Direct fluorescence microscopy of yeast wild-type cells expressing GFP-labeled Ump1 instead of the endogenous protein (top panel) and DAPI stained nuclei. (B) Fluorescence microscopy of GFP-labeled Ump1 in SRP1 wild type (top panel) and srp1-49 cells grown at permissive temperature (lower panel). The research in (A,B) is reprinted from Lehmann et al. (2002). (C) Fluorescence microscopy of GFP-tagged β5 in wild-type (top panel) and ump1Δ cells (lower panel). This research was originally published in EMBO reports (Lehmann et al., 2008). (D) Fluorescence microscopy of GFP-tagged Rpn11 in SRP1 wild-type (top panel) and srp1-49 cells grown at restrictive temperature (lower panel). (E) Fluorescence microscopy of GFP-tagged Rpn1 in SRP1 wild-type (top panel) and srp1-49 cells grown at restrictive temperature. The research in (D,E) was originally published in Wendler et al. (2004).

Figure 4

Proteasome localization during different growth phases in yeast. (A) In proliferating yeast, 15S precursor complexes, pre-holo-proteasomes, and 19S base complexes are imported into the nucleus via the cNLS pathway and importin αβ. Importin αβ binds to newly assembled proteasomal complexes in the cytoplasm. RanGTP dissociates the import complex in the nucleus, so that proteasome assembly and maturation can take place. (B) During quiescence, 26S proteasomes initially locate to the nucleoplasmic side of the NPC. Prolonged quiescence leads to the break up into 19S RP and 20S CP, which are sequestered by Blm10 to protein storage granules in the cytoplasm. (C) Upon resumption of growth after quiescence, Blm10 can act as nuclear transport receptor binding to 20S or 26S holo proteasomes. Blm10 import complexes are dissociated by RanGTP in the nucleus. The re-import of free 19S RP into the nucleus is not entirely understood.