Amino Acid Substitutions of Coiled-Coil Protein Tpr Abrogate Anchorage to the Nuclear Pore Complex but Not Parallel, In-Register Homodimerization

Abstract

Tpr is a protein component of nuclear pore complex (NPC)-attached intranuclear filaments. Secondary structure predictions suggest a bipartite structure, with a large N-terminal domain dominated by heptad repeats (HRs) typical for coiled-coil-forming proteins. Proposed functions for Tpr have included roles as a homo-or heteropolymeric architectural element of the nuclear interior. To gain insight into Tpr's ultrastructural properties, we have studied recombinant Tpr segments by circular dichroism spectroscopy, chemical cross-linking, and rotary shadowing electron microscopy. We show that polypeptides of the N-terminal domain homodimerize in vitro and represent ␣-helical molecules of extended rod-like shape. With the use of a yeast two-hybrid approach, arrangement of the coiled-coil is found to be in parallel and in register. To clarify whether Tpr can self-assemble further into homopolymeric filaments, the full-length protein and deletion mutants were overexpressed in human cells and then analyzed by confocal immunofluorescence microscopy, cell fractionation, and immuno-electron microscopy. Surplus Tpr, which does not bind to the NPC, remains in a soluble state of ϳ7.5 S and occasionally forms aggregates of entangled molecules but neither self-assembles into extended linear filaments nor stably binds to other intranuclear structures. Binding to the NPC is shown to depend on the integrity of individual HRs; amino acid substitutions within these HRs abrogate NPC binding and render the protein soluble but do not abolish Tpr's general ability to homodimerize. Possible contributions of Tpr to the structural organization of the nuclear periphery in somatic cells are discussed. INTRODUCTIONThe nuclear pore complex (NPC) is a highly complex structure of eightfold rotational symmetry that serves as the gateway for the exchange of cellular material between cytoplasm and nucleus in eukaryotes. Its core structure consists of central globular subunits flanked by a ring-like structure (annulus) at both the NPC's cytoplasmic (outer) and nucleoplasmic (inner) side. Both annuli are attachment sites for fibrils, also arranged in an eightfold symmetrical pattern but of distinctive shape and protein composition (for recent reviews, see Ohno et al., 1998;Stoffler et al., 1999;Ryan and Wente, 2000). Fibrils emanating from the outer annulus exist as short tufts of ϳ50 nm in length, whereas the rectilinear fibrils of 5-7 nm in diameter attached to the inner annulus vary in length. Approximately 60 -100 nm from the NPC's inner annulus, the intranuclear fibrils are laterally interconnected by another ring-like structure, sometimes described as the "terminal ring." Together, terminal ring and fibrils proximal to the NPC proper are considered to represent a structural and functional entity, called the nuclear "fishtrap" or "basket" (Ris, 1989(Ris, , 1991Jarnik and Aebi, 1991;Goldberg and Allen, 1992).Emanating from the terminal ring, additional fibrils project further into the nuclear interior. In amphibian oocytes these fibrils can be seve...

NUCLEAR PORE ANCHOR, theArabidopsisHomolog of Tpr/Mlp1/Mlp2/Megator, Is Involved in mRNA Export and SUMO Homeostasis and Affects Diverse Aspects of Plant Development

“…Nevertheless, on the light microscopy level, a continuous nuclear envelope staining was seen in several recent studies of human Tpr (Hase et al, 2001;Hase and Cordes, 2003;Krull et al, 2004). Similarly, in our system, we do not detect a punctate pattern for NUA.…”

Direct Interaction with Nup153 Mediates Binding of Tpr to the Periphery of the Nuclear Pore Complex

“…Rabbit and guinea-pig antibodies against amino acids (aa) 2063-2084 of human Tpr (hTpr) and aa 2285-2314 of hNup358/RanBP2 (Cordes et al, 1996(Cordes et al, , 1997, rabbit antibodies against aa 1-14 and aa 604 -615 of hTpr (Kuznetsov et al, 2002), mAb (mAb) 203-37 against hTpr (Cordes et al, 1997;Hase et al, 2001), mAb PF190x7A8 against an epitope located between aa 439 and 611 of rat Nup153 (Cordes et al, 1993), guinea-pig antibodies against aa 21-36 and 391-404 of hNup153 (Ferrando-May et al, 2001) and against murine p62 (Cordes et al, 1991), and mAb 9E10 for human c-Myc (ATCC CRL 1729) have been described. MAb R27 against mammalian lamin A and mAb X223 against B-type lamins (Hö ger et al, 1991) were kindly provided by G. Krohne.…”

“…These filamentous structures might be specific for oocytes, known to stockpile individual proteins in large quantities and various forms. In fact, it has been considered possible that they might represent a common stockpile of Tpr, Nup153, and perhaps even some other nucleoporins, later to be used during embryogenesis (Hase et al, 2001).…”

“…Tpr and Mlps are proteins forming coiled-coil dominated homodimers of extended rod-like shape (Kosova et al, 2000;Hase et al, 2001). Homodimerization occurs via the large N-terminal domain, which also includes a short sequence segment found to be essential for mediating binding of recombinant versions of Tpr to the NPC of transfected cells (Bangs et al, 1998;Cordes et al, 1998, Hase et al, 2001.…”

“…Homodimerization occurs via the large N-terminal domain, which also includes a short sequence segment found to be essential for mediating binding of recombinant versions of Tpr to the NPC of transfected cells (Bangs et al, 1998;Cordes et al, 1998, Hase et al, 2001. However, binding partners responsible for positioning Tpr at the nuclear periphery have remained elusive.…”