Domains of Gln3p Interacting with Karyopherins, Ure2p, and the Target of Rapamycin Protein

John Carvalho,X.F Steven Zheng

doi:10.1074/jbc.m300429200

Abstract

Gln3p is a GATA-type transcription factor responsive to the quality of nitrogen and carbon. In preferred nitrogen such as glutamine, Gln3p is phosphorylated and sequestered in the cytoplasm in a manner that is dependent on the target of rapamycin (TOR) protein and Ure2p. In nonpreferred nitrogen or nitrogen starvation, Gln3p is dephosphorylated and imported into the nucleus via karyopherin alpha/Srp1p. Upon reintroduction of preferred nitrogen, Gln3p is exported from the nucleus by Crm1p/Xpo1p. Although recent work has provided insights into Gln3p, a more detailed understanding is needed to elucidate the mechanism of its localization and function. In this study, we show that Gln3p contains canonical nuclear localization signal and nuclear export signal sequences necessary for its localization and interaction with its relevant karyopherins. In addition, we identify an N-terminal domain of Gln3p interacting with Ure2p and a C-terminal region for binding to TOR. Finally, we find a lysine/arginine-rich domain essential for the rapamycin-sensitive function, but dispensable for its localization. Our results reveal key domains of Gln3p important for its function and regulation.

Highlights

Nitrogen [6, 7]
We found that cells expressing a Gln3p-MYC9 mutant without the first 101 amino acids were still sensitive to rapamycin (Fig. 1, A and B)
A common feature of these rapamycin-resistant mutants is that the Gln3p transcriptional activation domain is deleted, suggesting that transcriptional activation is critical for the rapamycin-sensitive function of Gln3p

Summary

Introduction

It was recently shown that the target of rapamycin (TOR) protein-nitrogen (8 –11) and Snf1glucose signaling pathways regulate Gln3p [12]. Nitrogen starvation or inhibition of TOR by rapamycin causes rapid dephosphorylation and nuclear accumulation of Gln3p in vivo as well as expression of a wide range of NCR genes [8, 10, 21, 22]. Mutation in Srp1p blocks the nuclear import of Gln3p and the expression of Gln3p-dependent genes by rapamycin treatment or nitrogen starvation [11]. We localized a lysine/argininerich motif crucial for a rapamycin-sensitive function that is unrelated to Gln3p localization These results provide a detailed understanding of Gln3p structure and function

Methods

Results

Conclusion