The two forms of karyogamy transcription factor Kar4p are regulated by differential initiation of transcription, translation, and protein turnover

Abstract

Kar4p is a transcription factor in Saccharomyces cerevisiae that is required for the expression of karyogamy-specific genes during mating, for the efficient transit from G1 during mitosis, and for essential functions during meiosis. Kar4p exists in two forms: a constitutive slower-migrating form, which predominates during vegetative growth, and a faster-migrating form, which is highly induced by mating pheromone. Transcript mapping of KAR4 revealed that the constitutive mRNA was initiated upstream of two in-frame ATG initiation codons, while the major inducible mRNA originated between them. Thus, the two forms of Kar4p are derived from the translation of alternative transcripts, which possess different AUG initiation codons. Site-directed mutations were constructed to inactivate one or the other of the initiation codons, allowing the expression of the two Kar4p forms separately. At normal levels of expression, the constitutive form of Kar4p did not support wild-type levels of mating. However, the two forms of Kar4p could also be expressed separately from the regulatable GAL1 promoter, and no functional difference was detected when they were expressed at equivalent levels. Pulse-chase experiments showed that the induced form of Kar4p was highly expressed and stable during mating but rapidly turned over in vegetative cells. In contrast, the constitutively expressed longer form showed the same rate of turnover regardless of the growth condition. Furthermore, overexpression of either form of Kar4p in vegetative cells was toxic. Thus, the elaborate regulation of the two forms of Kar4p at the levels of transcription, translation, and protein turnover reflects the requirement for high levels of the protein during mating and for low levels during the subsequent phases of the cell cycle.

FIG. 1

Mapping of the 5′ mRNA start sites of KAR4. Two separate techniques, S1 nuclease protection and primer extension, were used to map the 5′ ends of KAR4 mRNA, as described in Materials and Methods. Total yeast RNA was isolated from a wild-type (WT) strain (MS1919) and an isogenic kar4Δ strain (MS3216) grown in the absence (−) or presence (+) of α-factor. Sequencing was performed with a WT KAR4 plasmid (pMR2516) as the template. The products from S1 nuclease protection, primer extension, and the sequencing were run in parallel on the same sequencing gel. The sequencing lanes are labeled in the order G A T C. The first (ATG₁) and second (ATG₂) translational start codons are indicated in the margin but appear on the sequencing gel as the complementary sequence TAC. The pheromone-induced transcriptional initiation sites are bracketed. Asterisks indicate the constitutively expressed transcriptional initiation sites.

FIG. 2

The separate expression of the two forms of Kar4p. (A) Expression patterns of the ATG mutants. The kar4Δ::HIS3 (MS3216) strain harbored either a wild-type (WT) KAR4 construct (pMR2654), the ATG₁→AAG construct (pMR3357), or the ATG₂→AAG construct (pMR3359). The cells were grown in the absence (−) or presence (+) of α-factor, as indicated. The 12CA5 monoclonal antibody was used to detect the triple HA epitope in the Kar4 proteins. The long form of Kar4::HAp migrates at 41.5 kDa, and the short form migrates at 38.5 kDa. (B) Differential expression of Kar4p by using a galactose-inducible promoter. Galactose induction of the P_GAL-KAR4::HA constructs in the presence (+) and absence (−) of pheromone (α-factor) is shown. A kar4Δ strain, MS3216, harbored the galactose-inducible promoter (P_GAL) constructs to produce the Kar4p-long (pMR3459) or the Kar4p-short (pMR3356) form. WT Kar4p (WT) expression from pMR2654 is shown as a reference for the mobility of the two forms. For this experiment, the induction conditions were 2% galactose plus 0.35% glucose for Kar4p-long and 2% galactose plus 0.2% glucose for Kar4p-short. For the controls, we confirmed that for strains carrying both constructs, Kar4p was not detected when the cells where not under galactose induction conditions (not shown).

FIG. 3

Modulation of Kar4p to sufficient levels for mating. (A) Western blot showing the modulation of Kar4p expression by glucose repression of galactose induction. A kar4Δ strain, MS3216, harbored the galactose-inducible promoter (P_GAL) constructs to produce the Kar4p-long (pMR3459) or the Kar4p-short (pMR3356) forms. Wild-type (WT) Kar4p expression from pMR2654 in the presence of α-factor (+αF) is shown as a reference. P_GAL induction was carried out in synthetic complete medium lacking uracil with 2% raffinose and 2% galactose and with various concentrations of glucose (% Glc). (B) Western blot showing comparable levels of Kar4p-long (long) and Kar4p-short (short) expression upon modulation of the galactose induction with glucose. For the strain harboring the Kar4p-long P_GAL construct, 2% galactose and 0.35% glucose were used. For the Kar4p-short P_GAL construct, 2% galactose and 0.2% glucose were used. (C) Both forms of Kar4p complement the mating defect of kar4Δ when fully expressed. Shown is a plate mating in which a MATa kar4Δ strain (MS3216) carried a plasmid expressing the wild-type KAR4 gene (pMR2654), a vector control (pRS416) (vector), or the galactose-inducible promoter constructs to produce Kar4p-long (pMR3459) (long) or Kar4p-short (pMR3356) (short). These strains were pregrown on synthetic complete plates lacking uracil with 2% raffinose and then mated to a MATα kar4-2150 lawn (MS2710) for 4 h on synthetic complete plates with 2% raffinose and 2% galactose. The mating plate was then replica printed to diploid-selective plates. (D) Mating efficiency decreases as Kar4p is repressed. The same strains as described for panel C were used. The left square is a key to the pattern of cells on the mating plates. The modulated mating assay is described in Materials and Methods. For the plates shown, the matings were conducted at glucose concentrations ranging from 0.1% to 0.6% to modulate the expression of Kar4p-long or Kar4p-short.

FIG. 4

Differential stabilities of the two Kar4p species. (A) Decay curves for Kar4p-short (diamonds) and Kar4p-long (squares). A KAR4::HA strain (MY5792) was pulsed for 10 min in the presence (open symbols) and absence (black symbols) of pheromone. The samples were chased for the times indicated, and the extracts were immunoprecipitated with anti-HA antibody as described in Materials and Methods. Kar4p bands were quantified, corrected for background, and normalized to the level at t = 0 min (100%) by processing with a PhosphorImager and ImageQuant software. The curves were fitted to the data points by exponential regression with Microsoft Excel software. (B) Two exposures of the pulse-chase gel in the presence of pheromone. The top panel is included to show the decay of Kar4p-short, and the bottom panel is included to show the decay of Kar4p-long. (C) The gel of the forms of Kar4p in the absence of pheromone.

FIG. 5

Vegetative growth defect caused by overexpression of Kar4p. Strains were streaked for single colony growth on 2% glucose (P_GAL repression) and 2% galactose (P_GAL induction). A wild-type strain (MY3375) carried either a plasmid expressing the wild-type (WT) KAR4 gene (pMR2654), a vector control (pRS416) (vector), or the galactose-inducible promoter construct to produce the Kar4p-long (pMR3459) or the Kar4p-short (pMR3356) form of Kar4p.