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The novel HECT-type ubiquitin-protein ligase Pub2p shares partially overlapping function with Pub1p in Schizosaccharomyces pombe

Department of Biology, Graduate School of Science, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558-8585, Japan

View larger version (64K): [in a new window]   Fig. 1. Structural comparison of Nedd4/Rsp5 family proteins. (A) Schematic representation of the domain structures. The C2, WW and HECT domains are indicated by shaded, hatched and black boxes, respectively. A.A., amino acids. (B) Amino-acid sequence alignment of the HECT domains. Amino acids that are identical in Pub2p are indicated by dark shading. The asterisk indicates the conserved cysteine residue (Cys639 for Pub2p), which is required for the thioester bond formation with the C-terminal glycine residue of ubiquitin. hNedd4 and mNedd4 indicate human and mouse Nedd4p, respectively. E6-AP is a prototype HECT-type ubiquitin-protein ligase from human. (C) A phylogenetic tree of HECT domains, constructed by the UPGMA method, of seven HECT-type ubiquitin ligases of S. pombe and Rsp5p of S. cerevisiae.

View larger version (76K): [in a new window]   Fig. 2. Transcriptional regulation of pub1+ and pub2+. (A) Northern analysis of pub1+ and pub2+ mRNA in mitotically growing cells. Synchronous cultures were attained by using the cdc25-22 temperature-sensitive mutant K164-9 (see the Materials and Methods). A 2.3 kb fragment containing pub1 and a 2.0 kb fragment containing pub2 were used as hybridization probes. The cdc22+ mRNA was also traced as an example whose levels fluctuate during cell cycle, peaking at the G1/S boundary (Hofmann et al., 1994). A 2.7 kb fragment containing cdc22 was used as a probe. The septation index, which reaches a peak in G1/S phase, was determined in order to monitor progression of the cell cycle. The agarose gels were stained with ethidium bromide, and ribosomal RNAs were used as loading controls and size markers. (B) Northern analysis of pub1+ and pub2+ mRNA after nitrogen starvation. h90 wild-type (L968) and ste11 (KJ33-1A) strains were cultured in EMM2-N liquid sporulation medium. The same probes as in (A) were used.

View larger version (39K): [in a new window]   Fig. 3. Growth behavior of pub1 and pub2 disruptants. (A) pH sensitivity of leucine auxotrophic pub+ (MM71-6B), pub1 (KKT81-3A) and pub2 (KKT85-8A) strains. These strains were grown for 5 days on EMM2 media at the indicated pH. (B) Colony formation at different incubation temperatures. L972 (wildtype), KKT83-9A (pub1), KKT39-7C (pub2) and KKT82-2D (pub1 pub2) were incubated on YEA medium at 30°C, 34°C, 35.5°C or 37°C.

View larger version (57K): [in a new window]   Fig. 4. Effects of overexpression of pub2+. (A) h- wild-type cells (MM72-11C) bearing pREP1-pub2-HA or pREP1-pub2CA-HA were grown up to mid-log phase in EMM2 containing 20 µM thiamine (lane 1, 2) and transferred to thiamine-free EMM2 to overexpress Pub2-HA and Pub2CA-HA (lane 3, 4). Western blotting was conducted with the anti-HA antibody (12CA5). (B) High levels of overexpression of pub2+ induces cell elongation. The MM72-11C strain was transformed with plasmid pREP1-pub2-HA (a and c) or pREP1-pub2CA-HA (b and d). Cells were grown up to mid-log phase in EMM2 containing 20 µM thiamine (a and b) and transferred to thiamine-free EMM2 (c and d). After incubation for 18 hours, cell size was observed under phase-contrast optics. Bar, 10 µm. (C) Cell multiplication after thiamine removal. MM72-11C cells transformed with either pREP1 (control) or pREP1-pub2+, were grown in EEM2 medium supplemented with 20 µM thiamine to mid-log phase and then transferred to EMM2 without thiamine to induce expression of pub2+. pub2+-overexpressing cells (closed squares) stopped growing after 16 hours, whereas control cells (open circles) continued to multiply. (D) Flow cytometric analysis for pub2+-overexpressing cells. Cells harboring pREP1-pub2+ or pREP1 were grown up to mid-log phase in EMM2 containing 20 µM thiamine and transferred to EMM2 without thiamine. Samples were withdrawn at the indicated time points. (E) Accumulation of Cdc25-6HA in pub2+-overexpressing cells. The OM1715 strain carrying either pREP1 (an empty vector), pREP1-pub2+ or pREP1-pub2CA was incubated in EMM2 without thiamine. Western blotting with anti-HA antibody (12CA5) revealed Cdc25-6HA. Tubulin was detected by anti--tubulin antibody (TAT-1) and was used as an internal reference marker.

View larger version (32K): [in a new window]   Fig. 7. The in vivo functional assay of a putative catalytic HECT domain of Pub2p. (A) Schematic representation of chimeric proteins of the Pub2 HECT domain fused with the N-terminal portion of Pub1p. `HECTPub2' and `HECT CAPub2' indicate the HECT domain of wild-type and the Cys639 Ala mutant Pub2p, respectively. (B) Complementation of the temperature-sensitive growth of pub1 by the chimeric proteins. pREP41-based plasmids carrying each chimeric construct were transformed into the KKT81-3A strain, which was grown on thiamine-free EMM2 plates at 37°C for 3 days.

View larger version (38K): [in a new window]   Fig. 5. Localization of Pub2p. (A) Localization of Pub1-GFP and Pub2-GFP. pREP81-based plasmids carrying GFP, Pub1-GFP or Pub2-GFP fusion constructs were introduced into the wild-type strain MM72-11C. After a 12 hour incubation on thiamine-free EMM2, cells were observed under a fluorescence microscope. (a) GFP, (b) Pub1-GFP and (c) Pub2-GFP. Scale bar, 10 µm. (B) Fractionation of cell lysates. Cell lysates were prepared from the Pub2-HA integrant (KKT87). P13 is a precipitate after centrifugation at 13,000 g, and P100 and S100 fractions are the sediment and the supernatant, respectively, after the second centrifugation at 100,000 g. Pub2-HA was detected by western blotting with anti-HA antibody 3F10.

View larger version (37K): [in a new window]   Fig. 6. Conjugation of Pub2p with ubiquitin. (A) A schematic representation of the fusion constructs. Ubiquitin (Ub) was fused to the C-terminus of glutathione-S-transferase (GST). A Myc epitope tag was fused to the C-terminus of the HECT domain of wild-type Pub2p (HECTPub2-Myc) or to that of Cys639 Ala mutant HECT domain (HECT CAPub2:Myc). (B) Association of the Pub2 HECT domain with ubiquitin. GST-Ub and HECT-Myc fusion proteins were co-expressed in wild-type cells (MM72-1D), which were incubated in thiamine-free EMM2 for 20 hours. GST-fusion protein was pulled down from crude cell-free extracts by glutathione beads. Pulled-down proteins were resolved by SDS-PAGE on 10% gels after treatment (+) or mock treatment (-) with 100 mM DTT. Western blots were probed with an anti-Myc antibody (9E10).