Figure 4.

NE Fusion in kar2-1 and prm3Δ crosses. (A) kar2-1 × wt. (1) Nuclear encounter of kar2-1 and wt, which express Nup49p-GFP (ATY1713 × ATY3359). (2) Transfer of HMG1-GFP from wt to kar2-1. Note the more uniform timing of arrival of signal in the trans-NE and trans-cortical ER than in wt. (2′) The parting of the apex of the cis-NE in the same preparation (ATY3197 × ATY1528). Unlike wt crosses, when HMG1-GFP arrives in the trans-NE of kar2-1, its intensity in the trans-ER is nearly equal to that of the trans-NE. Thus, arrival of HMG1-GFP in the trans-NE could be via the cortical ER in kar2-1 crosses. (3) To learn whether the inner membranes fuse in kar2-1× wt crosses, we have performed experiments in which one of the parental strains expresses GFP-Prm3p, Gar1p-GFP, or Nup49p-GFP (which would be expected to transfer only if both membranes fused). In each case, the kar2-1 cell expresses mRFP-HDEL. Crosses were examined after 3 h to follow GFP-Prm3p: panel a, ATY3197 × ATY3149; a nucleolar protein, Gar1p-GFP: panel b, ATY3197 × ATY3004; or tagged NPCs, Nup49p-GFP: panel c, ATY3197 × ATY1916. As shown, none are transferred. The partners that express Gar1p-GFP or Nup49p-GFP also express Spc42p-mRFP. ATY1916 additionally expresses Mid2p-GFP. GFP-Prm3p itself concentrates at the nexus (panel a). (B) prm3Δ× prm3Δ. (1) Encounter of two prm3Δ strains that express Nup49p-GFP (ATY3130 x ATY3131). (2) Transfer of HMG1-GFP (ATY3277 × ATY2782). Note that the intensity of the trans-NE signal generally exceeds that of the trans-cortical ER and note the focal gap. As in wt × wt crosses, during arrival of HMG1-GFP in the trans-cell, the intensity in the NE exceeds that of the cortical ER. (3) prm3Δ x prm3Δ crosses were examined after 3 h to learn whether nucleolar Gar1p-GFP (a: ATY3216 × ATY3298) or tagged NPCs (b: ATY3216 × ATY3131) are transferred. The MATa partner expresses mRFP-HDEL in both cases. Note the lack of transfer in each case.