Nucleoplasmic signals promote directed transmembrane protein import simultaneously via multiple channels of nuclear pores
SubjectActive Transport, Cell Nucleus
Fluorescence Recovery After Photobleaching
Nucleocytoplasmic Transport Proteins
Permanent link to this recordhttp://hdl.handle.net/20.500.12613/4479
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Abstract© 2020, The Author(s). 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.
Citation to related workSpringer Science and Business Media LLC
Has partNature Communications
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Quantifying Nucleoporin Stoichiometry Inside Single Nuclear Pore Complexes In vivoMi, L; Goryaynov, A; Lindquist, A; Rexach, M; Yang, W; Yang, Weidong|0000-0002-3554-3035 (2015-01-01)© 2015, Nature Publishing Group. All rights reserved. The nuclear pore complex (NPC) is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring-scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. NPCs are composed of ∼30 different nucleoporins (Nups), averaged at 8, 16 or 32 copies per NPC. This estimate has not been confirmed for individual NPCs in living cells due to the inherent difficulty of counting proteins inside single supramolecular complexes. Here we used single-molecule SPEED microscopy to directly count the copy-number of twenty-four different Nups within individual NPCs of live yeast, and found agreement as well as significant deviation from previous estimates. As expected, we counted 8 copies of four peripheral Nups and 16 copies of fourteen scaffold Nups. Unexpectedly, we counted a maximum of 16 copies of Nsp1 and Nic96, rather than 32 as previously estimated; and found only 10-15 copies of six other Nups, rather than 8 or 16 copies as expected. This in situ molecular-counting technology can test structure-function models of NPCs and other supramolecular structures in cells.
Unfoldomics of human diseases: Linking protein intrinsic disorder with diseasesUversky, VN; Oldfield, CJ; Midic, U; Xie, H; Xue, B; Vucetic, S; Iakoucheva, LM; Obradovic, Z; Keith, AK (2009-07-07)Background: Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) lack stable tertiary and/or secondary structure yet fulfills key biological functions. The recent recognition of IDPs and IDRs is leading to an entire field aimed at their systematic structural characterization and at determination of their mechanisms of action. Bioinformatics studies showed that IDPs and IDRs are highly abundant in different proteomes and carry out mostly regulatory functions related to molecular recognition and signal transduction. These activities complement the functions of structured proteins. IDPs and IDRs were shown to participate in both one-to-many and many-to-one signaling. Alternative splicing and posttranslational modifications are frequently used to tune the IDP functionality. Several individual IDPs were shown to be associated with human diseases, such as cancer, cardiovascular disease, amyloidoses, diabetes, neurodegenerative diseases, and others. This raises questions regarding the involvement of IDPs and IDRs in various diseases. Results: IDPs and IDRs were shown to be highly abundant in proteins associated with various human maladies. As the number of IDPs related to various diseases was found to be very large, the concepts of the disease-related unfoldome and unfoldomics were introduced. Novel bioinformatics tools were proposed to populate and characterize the disease-associated unfoldome. Structural characterization of the members of the disease-related unfoldome requires specialized experimental approaches. IDPs possess a number of unique structural and functional features that determine their broad involvement into the pathogenesis of various diseases. Conclusion: Proteins associated with various human diseases are enriched in intrinsic disorder. These disease-associated IDPs and IDRs are real, abundant, diversified, vital, and dynamic. These proteins and regions comprise the disease-related unfoldome, which covers a significant part of the human proteome. Profound association between intrinsic disorder and various human diseases is determined by a set of unique structural and functional characteristics of IDPs and IDRs. Unfoldomics of human diseases utilizes unrivaled bioinformatics and experimental techniques, paves the road for better understanding of human diseases, their pathogenesis and molecular mechanisms, and helps develop new strategies for the analysis of disease-related proteins. © 2009 Uversky et al; licensee BioMed Central Ltd.
A Spiroligomer α-Helix Mimic That Binds HDM2, Penetrates Human Cells and Stabilizes HDM2 in Cell CultureBrown, ZZ; Akula, K; Arzumanyan, A; Alleva, J; Jackson, M; Bichenkov, E; Sheffield, JB; Feitelson, MA; Schafmeister, CE (2012-10-18)We demonstrate functionalized spiroligomers that mimic the HDM2-bound conformation of the p53 activation domain. Spiroligomers are stereochemically defined, functionalized, spirocyclic monomers coupled through pairs of amide bonds to create spiro-ladder oligomers . Two series of spiroligomers were synthesized, one of structural analogs and one of stereochemical analogs, from which we identified compound 1, that binds HDM2 with a Kd value of 400 nM. The spiroligomer 1 penetrates human liver cancer cells through passive diffusion and in a dose-dependent and time-dependent manner increases the levels of HDM2 more than 30-fold in Huh7 cells in which the p53/HDM2 negative feed-back loop is inoperative. This is a biological effect that is not seen with the HDM2 ligand nutlin-3a. We propose that compound 1 modulates the levels of HDM2 by stabilizing it to proteolysis, allowing it to accumulate in the absence of a p53/HDM2 feedback loop. © 2012 Brown et al.