Cell Structure and Function

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YBR267w designated REI1 (required for isotropic bud growth) was isolated by two-hybrid screening using NIS1 encoding the neck protein as bait. Disruption of REI1 exhibited cold sensitive growth but did not exhibit a morphological defect. However, Δrei1Δnap1, Δrei1Δcla4 and Δrei1Δgin4 double disruptants exhibited an elongated cell morphology, which was suppressed by the disruption of SWE1, indicating that REI1 is a new member of genes belonging to the mitotic signaling network that negatively regulates Swe1 kinase. Δnap1 cells displayed a lower Gin4 kinase activity and a lower Gin4 protein level, both of which were recovered nearly to a wild type level in Δrei1Δnap1 cells. Interaction between Rei1 and Gin4 was suggested from our observation that Rei1 inhibited Gin4 kinase activity although weakly. The facts that although Δrei1Δnap1 cells displayed a severer elongated bud phenotype than Δnap1 cells, Gin4 kinase activity in Δrei1Δnap1 cells was higher than in Δnap1 cells, and that introduction of plasmid carrying a kinase inactive gin4 mutant gene into Δrei1Δgin4 cells suppressed their morphological defect, indicate that kinase activity of Gin4 is not required for isotropic bud growth. We found that Rei1 is localized to the cytoplasm throughout the cell cycle. In view of the fact that members belonging to the mitotic signaling network are localized to the bud neck, at least at some stage of the cell cycle, Rei1 is a unique component of this pathway.

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Here we report a new method of isolating epidermal desmosomes from Xenopus laevis, and a major constituent of desmosomes designated as Xenopus desmogleins (XDsg). Isolation of desmosomes from Xenopus laevis epidermis was carried out by a two step-incubation with different concentrations of NP-40. After discontinuous sucrose gradient centrifugation at 30,000 g for 60 min, a pure desmosomal fraction was obtained at 30%/40% interface. In the SDS-PAGE of isolated desmosomes, at least 12 bands (XDB1 to XDB12) were observed over a 75 kD region. Among them, three bands (XDB3, XDB7, XDB8; estimated MW 175, 124, and 112 kD respectively) were recognized as glycoproteins based on ConA binding. Monospecific polyclonal antibody against XDB3 cross-reacted with bovine Dsgs and vis-a-vis anti-bovine Dsgs with XDB3. By contrast, monospecific antibody against bovine Dsc a/b did not cross-react with either XDB7 or XDB8. Heterogeneous molecular constituents of desmosomal adhesion molecule, which have been observed among different bovine tissues, were confirmed in a phylogenetically different animal, Xenopus laevis. Combined results with other evidence could suggest an alternative system for desmosome-mediated cell adhesion.

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