Invertase activity in wild type (WT) and mutant strains grown under either de-repressing (A) or repressing (B) conditions. Error bars represent the standard error of the mean for four independent determinations.

Confocal images show localization of Mig1-GFP in either the presence (top panels) or absence (bottom panels) of glucose, in either wild type (WT) or mutant strains.

First lane (Input) shows the presence of the expressed proteins in the cell lysates; second lane (IP) shows the presence or absence of LexA-tagged proteins in the immunoprecipitated samples (top panel), and the immunoprecipitation of GFP-tagged Mig1 with the anti-GFP antibody in all the conditions tested (bottom panel). Vector, sample without any LexA-tagged protein.

(A) Quantitative fluorescent protein detection (QFPD) of Mig1-GFP in repressing conditions. Increasing amounts of protein from cytoplasmic, nuclear (perinuclear+lumenal), and perinuclear fractions isolated from wild type or mutant strains were loaded into microtiter wells (circles, left to right); fluorescence was measured as described in Materials & Methods. Units of invertase, also in repressing conditions, are shown for comparison. (B) Densitometric analysis of the data shown in A. The fraction of Mig1-GFP present in the cytoplasm (cytoplasmic; open bars), nuclear lumen (lumenal; shaded bars), and perinuclear compartment (perinuclear; filled bars) is shown for each strain. Error bars represent the standard error of the mean.

(A) Levels of HA-tagged Mig1 in crude lysate isolated from wild type, nup84Δ, nup120Δ, and nup133Δ cells grown in media containing glucose (repressing conditions) or pyruvate (derepressing conditions) as the carbon source. (B) Levels of GFP-tagged Mig1 in crude lysate isolated from wild type, nup120Δ, and nup133Δ cells grown in media containing glucose as the carbon source (repressing conditions). 100 µg total protein in each lane.

HA-tagged Mig1 (α-HA) was immunoprecipitated from wild type (A), nup84Δ (B), nup120Δ (C), and nup130Δ (D) cells grown in either the presence (+) or absence (−) of glucose. PCR was used to amplify the promoters of SUC2 and ACT1 (negative control) from immunoprecipitated material (α-HA), no antibody negative control (No Ab), and whole cell extracts (Input).

HA-tagged Mig1 (α-HA) was immunoprecipitated from wild type, nup120Δ, and nup130Δ cells grown in the presence of glucose. PCR was used to amplify the promoters of HXK1, HXT4, and TPS1 from immunoprecipitated material (α-HA), no antibody negative control (No Ab), and whole cell extracts (Input).

(A) Model 1, NPCs mark transcriptional boundaries and help regulate nucleosome position. NPCs interact with chromatin, establishing boundaries between active (green) and inactive (red) portions of the genome (represented by four loci on two DNA molecules attached to each nuclear pore). These boundaries provide a register from which the fine-scale positioning of nucleosomes can be established (nuclear pore on the left). By accumulating in the perinuclear subcompartment during growth on glucose, Mig1 can easily find its site immediately after SUC2 has visited the NPC and its promoter nucleosomes have been reset (nuclear pore on the right). In this model, deletion of either NUP120 or NUP133 disrupts nucleosome positioning throughout the genome, so that multiple Mig1 sites are masked and the repressor is blocked from binding DNA (not illustrated). (B) Model 2, NPCs facilitate DNA binding. (a) In the presence of glucose, Mig1 accumulates in the perinuclear subcompartment and SUC2 makes transient contact with NPCs. (b) Increased local concentration of both the promoter and the repressor facilitates Mig1 binding to its consensus site upstream of SUC2 and other target genes. (c) The repressed gene then moves back into the lumen, bound by Mig1. An alternative model not ruled out by the data presented here is that transient contact between Mig1 and the gene at NPCs is sufficient for repression. In this model, deletion of either NUP120 or NUP133 alters NPC structure in such a way that Mig1 can no longer associate, and thus can neither bind to DNA nor repress transcription from the promoters of glucose-repressed target genes. It should be noted that models (A) and (B) are not mutually exclusive.