Talk abstracts

Talk on Wednesday 03:00-03:15pm submitted by Mahesh Chemudupati

The nuclear export factor Gle1 is targeted to the nuclear envelope by two distinct cell cycle regulated mechanisms

Mahesh Chemudupati (Molecular Genetics, Ohio State University), Aysha H. Osmani (Molecular Genetics, Ohio State University), Stephen A. Osmani (Molecular Genetics, Ohio State University)

Abstract:
Our study aims to understand mitotic targeting of proteins to the nuclear envelope (NE). During mitosis in the model fungus Aspergillus nidulans, peripheral nuclear pore complex (NPC) proteins (Nups) disperse from NPCs thereby opening nuclear pores allowing nuclear entry of mitotic regulators. Paradoxically, a highly conserved peripheral Nup involved in nuclear export, Gle1, does not disperse from the NE during mitosis. We therefore asked whether Gle1 is tethered to the mitotic NE by other proteins. For this, we performed Gle1 affinity purifications and found that it co-purifies specifically with AN0162, which contains a C-terminal transmembrane domain. Endogenous GFP tagged AN0162 locates to the NE in a manner identical to Gle1, making AN0162 a candidate to tether Gle1 to the mitotic NE. Deletion experiments reveal that Gle1 associates with NPCs during interphase but transitions from NPCs to the INM through association with AN0162 during mitosis.

To identify structural features within AN0162 required for its INM targeting and Gle1 interaction, we replaced endogenous AN0162 with RFP-tagged truncated versions in a strain expressing endogenous tagged Gle1-GFP. This enabled tracking of the AN0162 truncations and how these truncations affected Gle1 targeting during the cell cycle. This approach identified two domains within AN0162 required for its INM targeting as well as a Gle1 binding domain (GBD). Thus strains expressing versions of AN0162 lacking its GBD targeted AN0162 normally throughout the cell cycle to the INM, but failed to target Gle1 to the mitotic INM. Conversely, strains lacking INM targeting domains of AN0162, but retaining the GBD, modified Gle1 distribution during mitosis. Our data reveals the existence of distinct and alternating cell cycle regulated mechanisms for NE protein targeting. We will next test if mitotic targeting of export factors is needed to maintain nuclear shape as nuclei of cells lacking AN0162 are rounder than wild type oblong nuclei.

References:
1. De Souza, C.P., et al., Partial nuclear pore complex disassembly during closed mitosis in Aspergillus nidulans. Curr Biol, 2004. 14(22): p. 1973-84.
2. De Souza, C.P. and S.A. Osmani, Double duty for nuclear proteins--the price of more open forms of mitosis. Trends Genet, 2009. 25(12): p. 545-54.
3. De Souza, C.P. and S.A. Osmani, Mitosis, not just open or closed. Eukaryot Cell, 2007. 6(9): p. 1521-7.

Keywords: mitosis, protein targeting, nucleus