Strain construction, materials, and Net1 mutagenesisAll strains used are in the W303 background (can1-100, leu2-3, his3-11, trp1-1, ura3-1, ade2-1) except where noted in the strain table (Supplementary Table 1). A strain expressing a stable form of Clb2 lacking both the KEN and destruction boxes (Clb2C 2 DK 100 )HA3 was used in over-expression experiments with Clb2 (1).Net1 mutant constructs were created as previously described (2). Briefly, a wild type NET1-myc9 epitope tagged construct was cloned into a modified pRS304 vector containing 300bp upstream of the ATG translation start site using NcoI and EagI. Sitedirected mutagenesis of Serine/Threonine to Alanine was carried out using QuikChange Site-Directed mutagenesis kit from Stratagene (La Jolla, CA). The indicated Serine/Threonine were mutated to Alanine in 169,212,231,252,259,356,362,384,385,497,611,676), 169,212,231,252,259),212,252), 166,212,252,297,304) where * indicates that residue 62 was mutated to ensure complete elimination of all Cdk consensus sites even though it was not determined to be phosphorylated in vivo. Mutagenesis was confirmed using restriction digests followed by Antibodies specifically reactive against the phosphopeptides were positively selected on a resin derivatized with the phosphopeptide immunogen and negatively selected by passage through a resin derivatized with the unphosphorylated version of the peptide. Antiphosphopeptide B (α-PP-B) was used in all experiments described since it generated the strongest signal against Phospho-Net1 ( Fig. 2A). Cell Growth and Synchronization ProceduresCells were grown in yeast extract-peptone (YP) or in yeast minimal (YM) media containing 2% glucose (YPD,YMD), 2% raffinose (YPR,YMR) or 2% galactose (YPG,YMG) as carbon source. Where appropriate, minimal media were supplemented with leucine, histidine, tryptophan, uracil, and adenine to complement auxotrophies.Synchronization of cells in G1 phase was achieved with α-factor added at 10 µg/ml for BAR1 cells and 0.1 µg/ml for bar1∆ cells for at least 3 hrs at 25°C. Cells were judged to be arrested when greater than 90% of cells displayed the elongated "shmoo" phenotype.Cells were released from α factor by filtration through a 0.2µm filter followed by a wash with 150 ml of YP, then resuspended in the desired volume at a density of 1 O.D. 600 /ml.For elutriation, cells were grown overnight in YP containing 2% raffinose and 2%galactose and harvested at log phase. Elutriation was performed as described (3-5) for the collection of small, unbudded G1 cells; contamination with budded cells was measured to be no more than 2%. For galactose induction experiments, cells were grown overnight in either YMR or YPR until an O.D. 600 of 1.0 was reached, then induced with 2% galactose followed by time point collection. Cell Extract Preparation and Western BlottingCells were grown to an O.D. 600 of 1.0, and for every time point 2 ml of culture was collected and TCA added to a final concentration of 20%. Cells were collected by centrifugation and washed with 2...

The budding yeast RENT complex, consisting of at least three proteins (Net1, Cdc14, Sir2), is anchored to the nucleolus by Net1. RENT controls mitotic exit, nucleolar silencing, and nucleolar localization of Nop1. Here, we report two new functions of Net1. First, Net1 directly binds Pol I and stimulates rRNA synthesis both in vitro and in vivo. Second, Net1 modulates nucleolar structure by regulating rDNA morphology and proper localization of multiple nucleolar antigens, including Pol I. Importantly, we show that the nucleolar and previously described cell cycle functions of the RENT complex can be uncoupled by a dominant mutant allele of CDC14. The independent functions of Net1 link a key event in the cell cycle to nucleolar processes that are fundamental to cell growth.