Mlp2p, A Component of Nuclear Pore Attached Intranuclear Filaments, Associates with Nic96p

Abstract

A fraction of the yeast nucleoporin Nic96p is localized at the terminal ring of the nuclear basket. When Nic96p was affinity purified from glutaraldehyde-treated spheroplasts, it was found to be associated with Mlp2p. Mlp2p, together with Mlp1p, are the yeast Tpr homologues, which form the nuclear pore-attached intranuclear filaments (Strambio-de-Castillia, C., Blobel, G., and Rout, M. P. (1999) J. Cell Biol. 144, 839 -855). Double disruption mutants of MLP1 and MLP2 are viable and apparently not impaired in nucleocytoplasmic transport. However, overproduction of MLP1 causes nuclear accumulation of poly(A)؉ RNA in a chromatin-free area of the nucleus.Nuclear pore complexes are complex structures within the nuclear membrane, which mediate nuclear import and export of transport substrates and shuttling receptors (1). Each nuclear pore complex (NPC) 1 consists of a basic framework (i.e. the ring/spoke complex) which exhibits an 8-fold symmetry and spans the double nuclear membrane (2-4). The NPC is attached to peripheral structures such as the short cytoplasmic filaments, the nuclear basket, the nuclear envelope lattice, pore-attached intranuclear filaments, and the nuclear lamina (5-13). Recently, some of these peripheral elements of the NPC gained increased attention since they were proposed to play an important role in the initial docking step of the transport substrate to the pores and the final release from the pores (8, 14 -19). In particular, the large nucleoporin Nup358, located at the tips of the short cytoplasmatically attached NPC filaments, is thought to mediate one of the first contacts with transport cargos via the importin/karyopherin ␣/␤ complex (15, 19 -21). On the other side of the nuclear envelope, release of the import cargo from the nuclear pores appears to occur at the terminal ring of the nuclear basket, and Nup153 might be involved in this final process (16,18,22). However, electron microscopy revealed that NPCs do not abruptly end at the nuclear basket, but are connected to a complicated meshwork of intranuclear structures. These structures consist of pore-attached filaments, which deeply penetrate into the nuclear interior or an underlying nuclear envelope lattice (6,7,11,(23)(24)(25)(26). Therefore, it could be hypothesized that nuclear transport does not end after release of the import cargo from the nuclear basket and followed by intranuclear diffusion, but instead the facilitated transport through the pore could continue on intranuclear "tracks" to distinct intranuclear sites. In a similar way, export cargos may use intranuclear filaments for transport from the nuclear interior to the NPCs. Accordingly, the "gene gating hypothesis" was presented to propose that nuclear pores are connected via intranuclear tracks to distinct locations inside the nucleus (27).The molecular composition of the intranuclear filamentous system (often referred to as the "nuclear skeleton" or "nuclear matrix"), which has been visualized by a variety of electron microscopy techniques is still poorly characte...

The C-terminal domain of myosin-like protein 1 (Mlp1p) is a docking site for heterogeneous nuclear ribonucleoproteins that are required for mRNA export

“…1E, lane 2). Srp1p was detected only in the unbound CT-Mlp1p fraction, indicating that these two proteins do not interact directly, as reported (23) (Fig. 1E, lanes 3 and 4).…”

“…1E). In this experiment, GST control protein or GST-CT-Mlp1p expressed in E. coli was bound to glutathione-Sepharose and incubated with either purified recombinant Nab2p or a control protein, Srp1p, which does not interact with Mlp1p (23). Results indicate that Nab2p binds directly to CT-Mlp1p, as indicated by the presence of Nab2p in the bound CT-Mlp1p fraction (Fig.…”

“…It has been suggested that the effect of Mlp1p on poly(A) RNA localization is mediated by hnRNPs (23). Here we present the identification of the hnRNPs, Nab2p and Npl3p, found in complex with Mlp1p.…”

“…Previous experiments have shown that overexpression of full-length Mlp1p causes accumulation of poly(A) RNA within the nucleus (23). Given the identification of a direct interaction between the CT-Mlp1p and Nab2p, an hnRNP, we hypothesized that overexpression of the CT-Mlp1p might block poly(A) RNA export by titrating and sequestering RNA-bound hnRNPs within the nucleus.…”

“…Both proteins are associated with the nuclear pore protein, Nic96p (23), and immuno-electron microscopy indicates that the proteins are localized to the nucleoplasmic side of the NPC with a distinct nuclear pool (21,23). Insight into the function of Mlp1p came from the observation that overexpression of Mlp1p causes poly(A) RNA accumulation within the nucleus, as well as aggregation of Mlp1p within a chromatin-free, non-nucleolar region of the nucleus (21,23). This phenotype has also been observed in mammalian cells on overexpression of Tpr (24).…”