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 (Ohio State Biochemistry Program and Department of Molecular Genetics), Aysha H. Osmani (Department of Molecular Genetics), Stephen A. Osmani (Ohio State Biochemistry Program and Department of Molecular Genetics)

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, one highly conserved peripheral Nup involved in nuclear export, Gle1, does not disperse from the NE during mitosis. Hence, we investigated how Gle1 is tethered to the mitotic NE. For this, we performed Gle1 affinity purifications and found that it co-purifies specifically with AN0162, which has a C-terminal transmembrane domain, suggesting AN0162 might tether Gle1 to the mitotic NE membrane. Deletion experiments revealed that Gle1 associates with NPCs during interphase but leaves them during mitosis to associate with AN0162 at the inner nuclear membrane (INM).

To identify structural features within AN0162 required for its INM targeting and Gle1 interaction, we replaced endogenous AN0162 with red fluorescent protein-tagged truncated versions in a strain expressing endogenous Gle1-GFP. This enabled tracking of the AN0162 truncations and their effect on Gle1 targeting during the cell cycle. This approach identified several domains within AN0162 required for its INM targeting including the C-terminal transmembrane domain and a domain sufficient for nuclear localization. A Gle1 binding domain (GBD) was also identified. Versions of AN0162 lacking its GBD were targeted to the INM normally but failed to target Gle1 to the INM during mitosis. Conversely, strains lacking INM targeting domains of AN0162, but retaining the GBD, modified mitotic Gle1 distribution. Our data therefore reveal the existence of distinct and alternating cell cycle regulated mechanisms for NE protein targeting. Interestingly cells lacking AN0162 have rounder nuclei, while cells overexpressing AN0162 have larger and highly misshapen nuclei. Thus AN0162 functions to target Gle1 to the INM only during mitosis and is important to maintain normal overall nuclear structure.

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, nuclear envelope