Mechanisms of Nucleocytoplasmic Transport Público
Lange, Allison (2008)
Published
Abstract
The genome of the eukaryotic cell is separated from the contents of the cytoplasm by the nuclear membrane. This division allows for precise regulation of cellular activity by providing controlled access to the nuclear interior; however, it also requires the existence of a highly specific mechanism of nuclear import to facilitate and manage this process. Accordingly, nuclear import factors recognize and bind protein cargo in the cytoplasm, traverse the nuclear membrane, release cargo into the nucleus, and return to the cytoplasm for further rounds of import. This dissertation focuses on the cargo recognition and release steps, while also delving into the state of import receptors when unbound by cargo. Proteins destined for the nucleus contain nuclear localization signals (NLSs), which are recognized by soluble receptors called importins or karyopherins. The classical bipartite NLS has traditionally been defined as consisting of two stretches of basic amino acids separated by a linker of 10 amino acids. In Chapter 2, we find that this canonical consensus is artificially limiting, and that classical bipartite NLSs with linker regions of between eight and twenty residues can mediate nuclear import and interaction with importin alpha. In Chapter 3, we investigate the recently proposed PY-NLS, which is recognized by KapBeta2 and essentially consists of a hydrophobic or basic region upstream of an R/H/K(X2-5)PY motif, and show for the first time that this new NLS is functional in vivo and conserved across species. In Chapter 4, we transition into the nuclear interior and examine the cargo release step by validating in vivo the structures of importin alpha with two factors proposed to be involved in the delivery of classical NLS-containing protein cargo: the export factor for importin alpha, Cse1/RanGTP, and the nucleoporin, Nup2. Finally, in Chapter 5, we contemplate the state of the import receptor once it has been recycled to the cytoplasm as it prepares for its next round of import. We solve the crystal structure of unbound importin beta and show that the receptor assumes a compact, ring-like conformation when not bound to cargo, similar to the free states of other characterized transport receptors.
Table of Contents
Chapter 1: Introduction and Background Overview of Nucleocytoplasmic Transport The Classical Nuclear Import Cycle The Classical Nuclear Localization Signal The Importin Alpha/cNLS Interaction Dissociation of cNLS-Cargo from Importin Alpha The Beta-Karyopherin Family of Receptors Demonstrating that an NLS is Functional in vivo Scope of this Dissertation Chapter 2: Redefining the Classical Nuclear Localization Signal (cNLS) and Exploring the Prevalence of cNLS-Containing Protein Cargo in Saccharomyces cerevisiae Introduction Experimental Procedures Results Discussion Chapter 3: A PY-NLS Nuclear Targeting Signal is Required for Nuclear Localization and Function of the Saccharomyces cerevisiae mRNA-Binding Protein, Hrp1 Introduction Experimental Procedures Results Discussion Chapter 4: The Role of Cse1 and Nup2 in Classical NLS-Cargo Release Introduction Experimental Procedures Results Discussion Chapter 5: A Structural and Functional Analysis of the Classical Import Receptor, Importin Beta: Getting to the Heart of Nuclear Transport Introduction Experimental Procedures Results Discussion Chapter 6: Conclusions and Discussion References
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