During the mating of yeast Saccharomyces cerevisiae, two haploid nuclei fuse to produce a diploid nucleus. This process requires the functions of BiP/Kar2p, a member of the Hsp70 family in the endoplasmic reticulum, and its partner protein, Jem1p. To investigate further the role of BiP and Jem1p in nuclear fusion, we screened for partner proteins for Jem1p by the yeast two-hybrid system and identified Nep98p. Nep98p is an essential integral membrane protein of the nuclear envelope and is enriched in the spindle pole body (SPB), the sole microtubule-organizing center in yeast. Temperature-sensitive nep98 mutant cells contain abnormal SPBs lacking the half-bridge, suggesting the essential role of Nep98p in the organization of the normal SPB. Additionally, nep98 mutant cells show defects in mitotic nuclear division and nuclear fusion during mating. Because Jem1p is not required for nuclear division, Nep98p probably has dual functions in Jem1p-dependent karyogamy and in Jem1p-independent nuclear division.In the sexual phase of yeast Saccharomyces cerevisiae, haploid cells of opposite mating types mate to produce diploid cells. After cell fusion, the two haploid nuclei fuse to form a diploid nucleus (1). This process, karyogamy, can be dissected into two steps: nuclear congression and nuclear fusion (1, 2). Analyses of yeast mutants defective in nuclear fusion have revealed the involvement of BiP/Kar2p, an Hsp70 molecular chaperone in the endoplasmic reticulum (ER) 1 (3). BiP also performs functions including protein import into the ER and ER-related protein degradation (4, 5). BiP has three functional partner proteins in the DnaJ family: Scj1p, Sec63p, and Jem1p (6 -8), and the functions of BiP are specified by different DnaJ-like proteins. Protein import into the ER lumen is mediated by BiP and Sec63p, and protein aggregation is prevented in the ER lumen by BiP with Scj1p and Jem1p (9). Nuclear fusion is facilitated by the Sec63p complex and Jem1p; the zygotes of kar2, sec63, and jem1 mutants contain two closely opposed haploid nuclei that do not fuse (2,8,10).As a step toward understanding the mechanism of nuclear membrane fusion by BiP with Jem1p, we have screened for proteins that interact with Jem1p using the yeast two-hybrid system. The identified protein, Nep98p, is an essential integral membrane protein of the nuclear envelope and is enriched in the spindle pole body (SPB), the sole microtubule-organizing center of budding yeast and a functional homologue of the centrosome in mammalian cells (11). The temperature-sensitive (ts) nep98 mutant cells have abnormal SPBs lacking the half-bridge and show defects in both mitotic nuclear division and karyogamy, suggesting that Nep98p is essential for SPB organization and function. ura3 leu2 trp1 his3 lys2 suc2) and SEY6211 (MATa ura3 leu2 trp1 his3 ade2 suc2) (12) were used as the parental strains for the mutants constructed in this study. SEY621D was constructed by mating SEY6210 with SEY6211. PJ69-4A (MATa ura3 leu2 trp1 his3 gal4 gal80 GAL2-ADE2 LYS2::GAL1-HIS3 me...

The epidermal growth factor receptor (EGFR) has an essential role in multiple signaling pathways, including cell proliferation and migration, through extracellular ligand binding and subsequent activation of its intracellular tyrosine kinase (TK) domain. The non-small cell lung cancer (NSCLC)-associated EGFR mutants, L858R and G719S, are constitutively active and oncogenic. They display sensitivity to TK inhibitors, including gefitinib and erlotinib. In contrast, the secondary mutation of the gatekeeper residue, T790M, reportedly confers inhibitor resistance on the oncogenic EGFR mutants. In this study, our biochemical analyses revealed that the introduction of the T790M mutation confers gefitinib resistance on the G719S mutant. The G719S/T790M double mutant has enhanced activity and retains high gefitinib-binding affinity. The T790M mutation increases the ATP affinity of the G719S mutant, explaining the acquired drug resistance of the double mutant. Structural analyses of the G719S/T790M double mutant, as well as the wild type and the G719S and L858R mutants, revealed that the T790M mutation stabilizes the hydrophobic spine of the active EGFR-TK conformation. The Met790 side chain of the G719S/T790M double mutant, in the apo form and gefitinib- and AMPPNP-bound forms, adopts different conformations that explain the accommodation of these ligands. In the L858R mutant structure, the active-site cleft is expanded by the repositioning of Phe723 within the P-loop. Notably, the introduction of the F723A mutation greatly enhanced the gefitinib sensitivity of the wild-type EGFR in vivo, supporting our hypothesis that the expansion of the active-site cleft results in enhanced gefitinib sensitivity. Taken together, our results provide a structural basis for the altered drug sensitivities caused by distinct NSCLC-associated EGFR mutations.

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