MOB1 sequence analysis. (A) MOB1 DNA sequence and ORF. Underlined sequences represent the donor, branch, and acceptor sites for mRNA splicing. Note that the branch site is noncanonical. The postion of the initiator methionine of an unspliced MOB1 is boxed. The arrow indicates the fusion junction in the two-hybrid prey plasmid. (B) Aligned sequences of a subset of class 1 Mob1p-related ORFs from various genome databases. Comparison of Mob1p (ScMob1) and Mob2p (ScMob2) to predicted amino acid sequences from A. thaliania (At#1 represents a contig of GenBank accession numbers H77164 and T04732; At#2, GenBank accession no. Z46539); Rice (GenBank accession no. C19722); human (Hs#1 represents a contig of GenBank accession numbers F11288, F11866, R19220, R13096, and R59435); mouse (Mm#1, GenBank accession no. AA118366); and C. elegans (Ce#1, GenBank accession no. Z69788; Ce#2, GenBank accession no. Z77660). Sequences shaded in black are identical. Sequences shaded in gray are conserved. X’s represent ambiguous sequence. Note that many of the sequences derive from expressed sequence tags (ESTs) and therefore may be incomplete. (C) Aligned sequences of a subset of Class 2 Mob1p-related ORFs from various genome databases. Mob1p (ScMob1) sequence is compared with predicted amino acid sequences of the class 2 Mob1p-related sequences isolated from mouse (Mm#2 represents a contig of GenBank accession numbers U01138, AA125007, and AA208960); human (Hs#2 represents a contig of GenBank accession numbers W76032, D59087, N55945, and T79518); C. elegans (Ce #3, GenBank accession no. L10990); C. briggsae (Cb, GenBank accession no. R03084); and S. masoni (Sm, GenBank accession no. AA1468).

Mob1p is a phosphoprotein. (A) Autoradiogram of precipitated ³²P-labeled GST-Mob1p from extracts of cells grown in the presence of [³²P]orthophosphate (see MATERIALS AND METHODS). Lane 1, ³²P-labeled GST-Mob1p precipitated by glutathione-Sepharose; lane 2, ³²P-labeled Mob1p separated from GST by thrombin cleavage; lane 3, remainder of ³²P-labeled protein on glutathione-Sepharose post-thrombin cleavage. (B) H6-Mob1p is phosphorylated by GST-Mps1p in vitro (see MATERIALS AND METHODS). Mps1p kinase assays of GST-Mps1p ± H6-Mob1p and ± MBP were separated by SDS-PAGE and processed for autoradiography. Positions of molecular weight markers are shown.

Cells harboring mob1 mutants arrest in late nuclear division. (A) Flow cytometric analysis of DNA content of a wild-type yeast strain grown at 25°C or 37°C for 3 h. The biphasic peaks represent cells with G1 (1N) and G2/M (2N) DNA content. (B) Flow cytometric analysis of DNA content of yeast harboring the mob1–77 allele grown at 25°C or 37°C for 3 h. At 37°C cells harboring mob1–77 mutation arrest as large budded cells with G2/M DNA content. Percent large-budded (LB) cells are noted. (C) Arrested mob1–77 cells (3 h at 37°C) were stained with anti-tubulin antibody (left panels) and 4,6-diamidino-2-phenylindole (right panels). The cells arrest with long mitotic spindles and separated chromatin masses. Three representative cells are shown.

Mad1p is hyperphosphorylated in mob1 mutants. Cells were synchronized in α-factor at 25°C and released in media containing 15 μg nocodozole (NZL) and 30 μg benomyl at 36°C. Samples were taken before (−) and after (+) treatment with nocododole and processed for 10% SDS PAGE and immunoblotted The immunoblot was probed with affinity- purified antiMad1p antibody. The lane loaded with extract from a strain deleted for MAD1 (mad1Δ) reveals an immunoreactive background protein. Lanes loaded with extract from wild type (WT), mob1–77, and mob1–95 cells are indicated. Mad1p hyperphosphorylation is detectable only in nocodazole-treated cells by the presence of slower migrating forms.

GST-Mob1p precipitates Mps1p protein kinase. (A) Expression and purification of GST-Mob1p from yeast (see MATERIALS AND METHODS). Lane 1, total yeast protein containing expressed GST-Mob1p stained with Coomassie brilliant blue on SDS-PAGE. Lane 2, GST-Mob1p purified from yeast extract on glutathione-Sepharose. Positions of molecular weight markers are shown. (B) Protein from yeast extract containing myc-epitope- tagged Mps1p was precipitated with anti-myc antibody (MYC IP), GST-Mob1p-bound Sepharose (GST-Mob1p), or GST-bound Sepharose (GST). CBB, Coomassie brilliant blue-stained gel of precipitants. Kinase assay, autoradiogram showing ³²P-labeled proteins in corresponding precipitants after performance of an Mps1p protein kinase assay. Immunoblot, precipitated protein were immunoblotted and probed with 9E10. The prominent ∼ 60 kDa band below myc-Mps1p on the immunoblot of MYC-IP is IgG heavy chain of the primary antibody recognized by the secondary antibody. Note that active Mps1p protein kinase is present in the anti-myc and GST-Mob1p precipitants but not in the GST precipitants. Also note that in addition to myc-Mps1p autophosphorylation, GST-Mob1p is phosphorylated in vitro. Molecular weight markers (M) and the positions of GST, GST-Mob1p, and myc-Mps1p are shown.

Positions of mob1 lesions on Mob1p. Amino acid substitutions in mob1 alleles are noted under wild-type Mob1p sequence. Amino acids represented in bold print are conserved among the class 1 Mob1p-like proteins (see Figure 1). Underlined sequences represent the amino acids changed in mob1 mutants.

MOB1 and MOB2 are required for the maintenance of ploidy. (A) Flow cytometric analysis of DNA content of yeast harboring various alleles of MOB1 grown at 25°C. Cells harboring mob1–77 or mob1–95 mutations contain haploid biphasic peaks representing G1 (1N) and G2/M (2N) DNA content. Cells harboring mob1–34, mob1–46, mob1–55, or mob1–83 mutations contain diploid biphasic peaks (2N and 4N) indicating that they have diploidized. Populations of yeast harboring mob1–67 mutation contain both haploid and diploid cells. (B) Flow cytometric analysis of DNA content of tetratype tetrad resulting from crossing mob1–95 and mps1–1 strains. Populations of wild-type cells and cells harboring mob1–95 or mps1–1 mutations are haploids containing both G1 (1N) and G2/M (2N) DNA content. Cells harboring both mob1–95 and mps1–1 have diploid DNA content (2N and 4N). (C) Flow cytometric analysis of DNA content of tetratype tetrad resulting from crossing mob2Δ and mps1–1 strains. Populations of wild-type cells and cells harboring mob2Δ or mps1–1 mutations are haploids containing both G1 (1N) and G2/M (2N) DNA content. Cells harboring both mob2Δ and mps1–1 have diploid DNA content (2N and 4N).