Determining the Architectures of Macromolecular Assemblies

Department of Bioengineering and Therapeutic Sciences, and California Institute for Quantitative Biosciences, Byers Hall, Suite 503B, 1700 4th Street, University of California at San Francisco, San Francisco, California 94158-2330, USA.
Nature (Impact Factor: 41.46). 12/2007; 450(7170):683-94. DOI: 10.1038/nature06404
Source: PubMed


To understand the workings of a living cell, we need to know the architectures of its macromolecular assemblies. Here we show how proteomic data can be used to determine such structures. The process involves the collection of sufficient and diverse high-quality data, translation of these data into spatial restraints, and an optimization that uses the restraints to generate an ensemble of structures consistent with the data. Analysis of the ensemble produces a detailed architectural map of the assembly. We developed our approach on a challenging model system, the nuclear pore complex (NPC). The NPC acts as a dynamic barrier, controlling access to and from the nucleus, and in yeast is a 50 MDa assembly of 456 proteins. The resulting structure, presented in an accompanying paper, reveals the configuration of the proteins in the NPC, providing insights into its evolution and architectural principles. The present approach should be applicable to many other macromolecular assemblies.

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Available from: Michael P Rout
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    • "NPC assembly during interphase requires the local fusion of the inner and outer nuclear membranes (INM and ONM) to form a pore in the NE (Fernandez-Martinez and Rout, 2009); fusion is coupled to the recruitment of $30 distinct NPC constituents (nucleoporins or nups). Because nups are found in multiple copies in a mature NPC, NPC assembly requires at least 450 nup protomers in yeast (Alber et al., 2007a, 2007b) and many more in human cells (Bui et al., 2013). The spatiotemporal regulation of these events and the biochemical intermediates in this process remain illdefined , although integral INM proteins of the LEM (Lap2- emerin-MAN1) (Yewdell et al., 2011) and SUN (Sad1, UNC84) families (Talamas and Hetzer, 2011) might help form early NPC assembly intermediates. "
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    ABSTRACT: The maintenance of nuclear compartmentalization by the nuclear envelope and nuclear pore complexes (NPCs) is essential for cell function; loss of compartmentalization is associated with cancers, laminopathies, and aging. We uncovered a pathway that surveils NPC assembly intermediates to promote the formation of functional NPCs. Surveillance is mediated by Heh2, a member of the LEM (Lap2-emerin-MAN1) family of integral inner nuclear membrane proteins, which binds to an early NPC assembly intermediate, but not to mature NPCs. Heh2 recruits the endosomal sorting complex required for transport (ESCRT)-III subunit Snf7 and the AAA-ATPase Vps4 to destabilize and clear defective NPC assembly intermediates. When surveillance or clearance is compromised, malformed NPCs accumulate in a storage of improperly assembled nuclear pore complexes compartment, or SINC. The SINC is retained in old mothers to prevent loss of daughter lifespan, highlighting a continuum of mechanisms to ensure nuclear compartmentalization.
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    • "Proteomic and microscopic analysis have deciphered that the entire NPC is comprised of distinct subcomplexes that are octagonally arranged around a central channel (Cronshaw et al., 2002; Alber et al., 2007a, b; Fiserova et al., 2009; Degrasse and Devos, 2010; Tamura et al., 2010). The size of a single NPC varies between ~60 MDa in yeast and ~120 MDa in metazoans but contain similar sets of core proteins. "
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