Many nuclear-targeted proteins are transported through the nuclear pore complex (NPC) by the importin-alpha:beta receptor. We now show that Npap60 (also called Nup50), a protein previously believed to be a structural component of the NPC, is a Ran binding protein and a cofactor for importin-alpha:beta-mediated import. Npap60 is a tri-stable switch that alternates between binding modes. The C terminus binds importin-beta through RanGTP. The N terminus binds the C terminus of importin-alpha, while a central domain binds importin-beta. Npap60:importin-alpha:beta binds cargo and can stimulate nuclear import. Endogenous Npap60 can shuttle and is accessible from the cytoplasmic side of the nuclear envelope. These results identify Npap60 as a cofactor for importin-alpha:beta nuclear import and as a previously unidentified subunit of the importin complex.
Ubiquitin is a small polypeptide that is conjugated to proteins and commonly serves as a degradation signal. The attachment of ubiquitin (Ub) to a substrate proceeds through a multi-enzyme cascade involving an activating enzyme (E1), a conjugating enzyme (E2), and a protein ligase (E3). We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11. We now show that the import mechanism for UbcM2 and two other human class III E2s (UbcH6 and UBE2E2) uniquely requires the covalent attachment of Ub to the active site cysteine of these enzymes. This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes. Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.
The Nrf2 transcription factor is a master regulator of the cellular anti-stress response. A population of the transcription factor associates with the mitochondria through a complex with KEAP1 and the mitochondrial outer membrane histidine phosphatase, PGAM5. To determine the function of this mitochondrial complex, we knocked down each component and assessed mitochondrial morphology and distribution. We discovered that depletion of Nrf2 or PGAM5, but not KEAP1, inhibits mitochondrial retrograde trafficking induced by proteasome inhibition. Mechanistically, this disrupted motility results from aberrant degradation of Miro2, a mitochondrial GTPase that links mitochondria to microtubules. Rescue experiments demonstrate that this Miro2 degradation involves the KEAP1-cullin-3 E3 ubiquitin ligase and the proteasome. These data are consistent with a model in which an intact complex of PGAM5-KEAP1-Nrf2 preserves mitochondrial motility by suppressing dominant-negative KEAP1 activity. These data further provide a mechanistic explanation for how age-dependent declines in Nrf2 expression impact mitochondrial motility and induce functional deficits commonly linked to neurodegeneration.
Summary Human Papillomaviruses (HPV) 16 is a DNA virus encoding three oncogenes – E5, E6, and E7. The E6 and E7 proteins have well-established roles as inhibitors of tumor suppression, but the contribution of E5 to malignant transformation is controversial. Using spontaneously immortalized human keratinocytes (HaCaT cells), we demonstrate that expression of HPV16 E5 is necessary and sufficient for the formation of bi-nucleated cells, a common characteristic of precancerous cervical lesions. Expression of E5 from non-carcinogenic HPV6b does not produce bi-nucleate cells. Video microscopy and biochemical analyses reveal that bi-nucleates arise through cell-cell fusion. Although most E5-induced bi-nucleates fail to propagate, co-expression of HPV16 E6/E7 enhances the proliferation of these cells. Expression of HPV16 E6/E7 also increases bi-nucleated cell colony formation. These findings identify a new role for HPV16 E5 and support a model in which complementary roles of the HPV16 oncogenes lead to the induction of carcinogenesis.
The Ran binding protein RanBP1 is localized to the cytosol of interphase cells. A leucine-rich nuclear export signal (NES) near the C terminus of RanBP1 is essential to maintain this distribution. We now show that RanBP1 accumulates in nuclei of cells treated with the export inhibitor, leptomycin B, and collapse of the nucleocytoplasmic Ran:GTP gradient leads to equilibration of RanBP1 across the nuclear envelope. Low temperature prevents nuclear accumulation of RanBP1, suggesting that import does not occur via simple diffusion. Glutathione S-transferase (GST)-RanBP1(1-161), which lacks the NES, accumulates in the nucleus after cytoplasmic microinjection. In permeabilized cells, nuclear accumulation of GST-RanBP1(1-161) requires nuclear Ran:GTP but is not inhibited by a dominant interfering G19V mutant of Ran. Nuclear accumulation is enhanced by addition of exogenous karyopherins/importins or RCC1, both of which also enhance nuclear Ran accumulation. Import correlates with Ran concentration. Remarkably, an E37K mutant of RanBP1 does not import into the nuclei under any conditions tested despite the fact that it can form a ternary complex with Ran and importin . These data indicate that RanBP1 translocates through the pores by an active, nonclassical mechanism and requires Ran:GTP for nuclear accumulation. Shuttling of RanBP1 may function to clear nuclear pores of Ran:GTP, to prevent premature release of import cargo from transport receptors.The defining feature of eukaryotic cells is the compartmentalization of DNA replication and transcription within the nucleus. Access to the nuclear compartment is provided by pores that are plugged through the double membrane of the nuclear envelope (for reviews, see references 15 and 66). A multitude of soluble transport receptors and accessory proteins controls the transit of protein and nucleic acid cargo through these pores (for recent reviews, see references 24, 40, 42, and 64). The vectoriality of cargo transport is in many cases dependent on the asymmetric distribution of Ran, a small GTP binding protein, and of its regulatory factors (8,17). Ran is predominantly nuclear and is believed to be maintained in the GTPbound state (Ran:GTP) by a guanine nucleotide exchange factor, RCC1, which is associated with chromatin in the nucleus (7, 58). A Ran-specific GTPase-activating protein, Ran-GAP, which converts Ran to the GDP-bound state, is excluded from the nucleus (5,13,30). This asymmetric arrangement of regulatory factors ensures that a steep Ran:GTP gradient exists across the nuclear pores, and the collapse of this gradient inhibits many forms of nuclear traffic (32, 52).
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