Structural basis for leucine-rich nuclear export signal recognition by CRM1

Dong, Xinhong; Biswas, Ashim Kumar; Süel, Katherine E.; Jackson, Lindsey N.; Martinez, Rita; Gu, Hongmei; Chook, Yuh Min

doi:10.1038/nature07975

Cited by 294 publications

(410 citation statements)

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“…Such an export mechanism has been identified, for example, in Snurportin-1. Snurportin-1 binds CRM1 in a bipartite manner, with a leucine-rich NES located in the N terminus and a second signal in a basic surface on the nucleotide binding domain (12). The second possibility is that CANCp2 and/or the full-length Gag protein forms or exposes one or more noncanonical determinants that are not formed by individually expressed MA, CA, or NCp2.…”

Section: Discussionmentioning

confidence: 99%

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Kemler

Saenz

Poeschla

2012

J Virol

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Lentiviral genomic RNAs are encapsidated by the viral Gag protein during virion assembly. The intracellular location of the initial Gag-RNA interaction is unknown. We previously observed feline immunodeficiency virus (FIV) Gag accumulating at the nuclear envelope during live-cell imaging, which suggested that trafficking of human immunodeficiency virus type 1 (HIV-1) and FIV Gag may differ. Here we analyzed the nucleocytoplasmic transport properties of both Gag proteins. We discovered that inhibition of the CRM1 nuclear export pathway with leptomycin B causes FIV Gag but not HIV-1 Gag to accumulate in the nucleus. Virtually all FIV Gag rapidly became intranuclear when the CRM1 export pathway was blocked, implying that most if not all FIV Gag normally undergoes nuclear cycling. In FIV-infected feline cells, some intranuclear Gag was detected in the steady state without leptomycin B treatment. When expressed individually, the FIV matrix (MA), capsid (CA), and nucleocapsid-p2 (NC-p2) domains were not capable of mediating leptomycin B-sensitive nuclear export of a fluorescent protein. In contrast, CA-NC-p2 did mediate nuclear export, with MA being dispensable. We conclude that HIV-1 and FIV Gag differ strikingly in a key intracellular trafficking property. FIV Gag is a nuclear shuttling protein that utilizes the CRM1 nuclear export pathway, while HIV-1 Gag is excluded from the nucleus. These findings expand the spectrum of lentiviral Gag behaviors and raise the possibility that FIV genome encapsidation may initiate in the nucleus.G ag is both necessary and sufficient for retrovirus particle formation and budding. The protein is translated on free polysomes and targeted to the plasma membrane, where virus particles are assembled (16). A central question is whether Gag and the viral genomic RNA (gRNA) associate at the plasma membrane or earlier during assembly and, if so, whether this occurs in the cytosol; in association with organelles, e.g., cotranslationally; or even in the nucleus, as was described previously for an alpharetrovirus (17). Human immunodeficiency virus type 1 (HIV-1) gRNAs become anchored at the plasma membrane before particle assembly is detectable, but it is not clear whether the Gag-gRNA complexes of this and other lentiviruses first form in the cytoplasm and then transit to the plasma membrane or the gRNA traffics there independently (26,27). Biochemical experiments supported the former scenario, with a monomer or low-order multimers forming on HIV-1 gRNAs and higher-order multimer formation depending on subsequent plasma membrane interactions (33).Gag is encoded by the full-length unspliced viral RNA. To circumvent the cellular checkpoint that prevents the export of intron-containing mRNA, lentiviruses express Rev, which functions as an adaptor between the cellular karyopherin CRM1/exportin-1 and a Rev response element (RRE) located in the 3= region of unspliced viral RNAs (9). However, the main cellular function of the CRM1 nuclear export pathway is the export of cellular proteins containing a...

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Section: Discussionmentioning

confidence: 99%

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Kemler

Saenz

Poeschla

2012

J Virol

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“…by guest www.bloodjournal.org From RAN or cargo protein alone is weak, simultaneous binding of RAN and cargo to XPO1 increases its affinity to both by 1000-fold. 24,34 XPO1/RAN-mediated export is increased in a variety of cancers. Mechanistically, overexpression of XPO1 enhances export of nuclear tumor suppressor proteins such as p53, BRCA1, allophycocyanin, and NMP1, resulting in drug resistance.…”

Section: Discussionmentioning

confidence: 99%

shRNA library screening identifies nucleocytoplasmic transport as a mediator of BCR-ABL1 kinase-independent resistance

Khorashad

Eiring

Mason

et al. 2015

Blood

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The mechanisms underlying tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML) patients lacking explanatory BCR-ABL1 kinase domain mutations are incompletely understood. To identify mechanisms of TKI resistance that are independent of BCR-ABL1 kinase activity, we introduced a lentiviral short hairpin RNA (shRNA) library targeting ∼5000 cell signaling genes into K562 R , a CML cell line with BCR-ABL1 kinaseindependent TKI resistance expressing exclusively native BCR-ABL1. A customized algorithm identified genes whose shRNA-mediated knockdown markedly impaired growth of K562 R cells compared with TKI-sensitive controls. Among the top candidates were 2 components of the nucleocytoplasmic transport complex, RAN and XPO1 (CRM1). shRNA-mediated RAN inhibition or treatment of cells with the XPO1 inhibitor, KPT-330 (Selinexor), increased the imatinib sensitivity of CML cell lines with kinase-independent TKI resistance. Inhibition of either RAN or XPO1 impaired colony formation of CD34 1 cells from newly diagnosed and TKI-resistant CML patients in the presence of imatinib, without effects on CD34 1 cells from normal cord blood or from a patient harboring the BCR-ABL1 T315I mutant. These data implicate RAN in BCR-ABL1 kinase-independent imatinib resistance and show that shRNA library screens are useful to identify alternative pathways critical to drug resistance in CML. (Blood. 2015;125(11):1772-1781

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“…This consensus sequence is found in PP32 LRRs 1-4, at least one of which is likely to interact with Crm1. Structures of Crm1 complexes have shown that the conserved Φ's of the consensus sequence dock into the pockets of the hydrophobic cleft of Crm1 (58,59). Because the conserved Φ's of PP32 make up its core and are not solvent-exposed, binding to Crm1 is likely to require partial unfolding of PP32.…”

Section: Possible Determinants For Folding Pathway Selections In Othementioning

confidence: 99%

Highly polarized C-terminal transition state of the leucine-rich repeat domain of PP32 is governed by local stability

Dao

Majumdar

Barrick

2015

Proc. Natl. Acad. Sci. U.S.A.

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The leucine-rich repeat domain of PP32 is composed of five β-strand-containing repeats anchored by terminal caps. These repeats differ in sequence but are similar in structure, providing a means to connect topology, sequence, and folding pathway selection. Through kinetic studies of PP32, we find folding to be ratelimited by the formation of an on-pathway intermediate. Destabilizing core substitutions reveal a transition state ensemble that is highly polarized toward the C-terminal repeat and cap. To determine if this nucleus for folding corresponds to the most stable region of PP32, we monitored amide hydrogen exchange by NMR spectroscopy. Indeed, we find the highest protection to be biased toward the C terminus. Sequence manipulations that destabilize the C terminus spread out the transition state toward the middle of the protein. Consistent with results for helical ankyrin repeat proteins, these results suggest that local stabilities determine folding pathways.A lmost 50 y ago, Cyrus Levinthal suggested that protein folding is a guided process, rather than a random search (1). Since then, it has been proposed that rapid folding can be accomplished via a single preferred pathway (2-4). However, contrasting models of funneled, nonspecific folding capture key aspects of the folding process, including specific intermediates and transient stable structures (5, 6). To what extent does protein folding follow a single pathway, and to the extent that a single pathway dominates, what determines that pathway?For some globular proteins, specific kinetic intermediates have been identified with features of thermodynamically stable substructures of the native state. This observation suggests specific pathways that correspond to low energy folding routes (7-12). However, directly testing this thermodynamic control of folding pathway selections requires mapping of local stability. Hydrogen exchange methods have provided the most detailed energy maps of native proteins (13), but sequence-distant contacts and irregular tertiary structures of globular proteins tend to blur the boundaries of local stability.In contrast to globular proteins, elongated repeat proteins comprise tandem repeated secondary structural units, giving rise to regular topology lacking sequence-distant contacts. As a result, the contributions made by different regions of a repeat protein to its folding thermodynamics and kinetics can be easily dissected and compared. If local stability dictates folding, a preferred pathway should be observed, because repeats differ in sequence. If topology drives folding, multiple parallel pathways would be observed, because the repeats are similar in structure. For designed consensus α-helical ankyrin repeat constructs, with repeats of nearly identical sequence, folding proceeds via parallel pathways (14). In contrast, naturally occurring ankyrin repeat proteins with repeats of high sequence variation fold through preferred pathways (15-18). Regions that initiate folding correspond to low-energy structures, based on energy ...

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Structural basis for leucine-rich nuclear export signal recognition by CRM1

Cited by 294 publications

References 50 publications

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

shRNA library screening identifies nucleocytoplasmic transport as a mediator of BCR-ABL1 kinase-independent resistance

Highly polarized C-terminal transition state of the leucine-rich repeat domain of PP32 is governed by local stability

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