Identification and Characterization of Mlp1p and Mlp2p : Molecular Components of Filaments Localized at the Interface between the Nuclear Pore Complex and Nuclear Interior

Author

Caterina Strambio-De-Castillia

Date of Award

1998

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Blobel Laboratory

Abstract

In eukaryotic cells the segregation of the genome in a closed organelle requires an efficient mechanism to ensure the constant exchange of material between the nucleus and the cytoplasm. Nuclear pore complexes (NPCs) provide the only known sites for exchange of material across the nuclear envelope (NE). A wealth of evidence has accumulated throughout the years that strongly suggest that the NPCs are structurally linked to the nuclear interior. It has long been proposed that this structural continuity is essential for the efficient exchange of material between the nuclear interior and the cytoplasm. Unfortunately, the understanding of the molecular basis of such functional and structural connections has lagged behind. This dissertation describes a combined biochemical and immunological approach aimed at the identification of novel yeast proteins that could be involved in providing such link. The first step of this approach was the development of a large scale enrichment procedure to prepare yeast nuclear envelopes (NEs). These NEs can be stripped of peripheral proteins to produce a heparin-extracted NE (H-NE) fraction highly enriched in integral membrane proteins. Extraction of H-NEs with detergents revealed previously uncharacterized ring structures associated with the NE that apparently stabilize the grommets of the nuclear pore complexes (NPCs). The high yields obtained throughout the fractionation procedure allowed balance-sheet tabulation of the subcellular distribution of various NE and non-NE associated proteins. As the second step of the approach described here, three different highly enriched NE-derived fractions were used to generate a panel of 114 monoclonal antibodies (mAbs) against NE-associated antigens. Finally, this panel of anti-NE mAbs were subjected to a novel NPC-clustering screen aimed at the identification of NE-associated antigens that were only peripherally associated with the NPC. Two mAbs were isolated using this screen (MAb148G11 and MAb215B9). Both of these mAbs were found to recognize the same -220 kD protein (p220) on immunoblots of highly enriched NE fractions. The gene encoding p220 was cloned and was found to be the previously identified gene of unknown function, MLP1. Disruptions of MLP1 and its homologue MLP2 (the uncharacterized yeast ORF, YIL149C), were found to be non lethal either separately or in combination. Though both M lplp and Mlp2p largely cofractionated with isolated NEs, neither cofractionated with isolated NPCs. Ultrastructural localization demonstrated that both Mlplp and Mlp2p are localized to filaments that appear to connect the NPC to the nuclear interior. Functional studies performed using yeast strains harboring a double deletion of MLP1 and MLP2 suggested that these proteins could be involved in facilitating nuclear import and led to the proposal of a model for the possible role of these proteins in nuclear transport.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Recommended Citation

Strambio-De-Castillia, Caterina, "Identification and Characterization of Mlp1p and Mlp2p : Molecular Components of Filaments Localized at the Interface between the Nuclear Pore Complex and Nuclear Interior" (1998). Student Theses and Dissertations. 377.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/377