Figure 2.

The Nup1/Nup60 N Termini Are Sufficient to Associate with NPCs

(A) A plasmid-borne NUP1 (1-123)-TAP construct (GAL promoter) was transformed into wild-type cells and tandem-affinity-purified following galactose-induced overexpression (4 hr). The calmodulin eluate was analyzed by SDS-PAGE and Coomassie staining. Assigned subunits were determined by mass spectrometry (roman numerals refer to cut-out bands, see Figure S2B). Asterisks indicate degradation products of endogenous full-length Nup1. Ssa1/Ssb1 are Hsp70-type chaperones, Kar2 is an ATPase involved in ER protein import.

(B) Live imaging of NUP188-GFP nup133Δ cells expressing the indicated plasmid-borne NUP1 fragments (GAL promoter). Cells were grown in raffinose media to obtain low levels of Nup1 expression sufficient for depiction. Scale bar represents 2 μm.

(C) Live imaging of wild-type and nup133Δ cells expressing the indicated NUP60 constructs (GAL promoter). As in (B), low level protein expression was achieved by growth in raffinose medium. Abnormally shaped nuclei were observed in nup133Δ cells expressing Nup60 (1-47). NLS, SV40 large T-antigen NLS. Scale bar represents 3 μm.

(D) Coomassie-stained gel of a Nup60 (1-162) fragment purified and analyzed as in (A). Assigned subunits were determined by mass spectrometry (roman numerals refer to cut out bands, see Figure S2D). Asterisk indicates degradation products of the bait protein. Sse1/Ssc1/Ssa1 are chaperones.

(E) Analysis of Nup60 (1-47) localization in kap123Δ NUP188-GFP cells. Cells were grown in raffinose as in (B) and (C). Also see Figure S2E. Scale bar represents 3 μm.