Figure 1.
N. crassa SIN components are required for septum formation but display distinct mutant characteristics
(A) Deletion strains defective in the indicated SIN components generate thin and aseptate hypha in young colonies (18 h time point). In older colonies, the septation defects were suppressed in ∆sid-1 and ∆cdc-14 strains (36 h time point). Cell wall and septa were labeled with Calcofluor White. (B) SIN mutants showed cytoplasmic leakage (magnified inserts), but, due to the fast ability to septate, ∆sid-1 and ∆cdc-14 generated abundant aerial mycelium and asexual spores (conidia; plate morphology). (C) SIN mutants displayed distinct abnormalities during sexual development. wt x ∆ crosses with ∆cdc-7(het) and ∆dbf-2(het) resulted in the frequent formation of large, banana-shaped ascospores. In contrast, wt x ∆sid-1(het) progeny morphology was normal, while crosses of wt x ∆cdc-14(het) produced no mature perithecia .
Figure 2.
N. crassa SIN components localize to SPBs and septa (A)
Functional GFP fusion proteins of CDC-7, SID-1, CDC-14 and DBF-2 localized to spindle pole bodies (arrows) and as constricting rings at forming septa. Nuclei were labeled with histone H1-RFP, the cell wall was stained with Calcofluor White. (B) The localization of the three SIN kinases CDC-7, SID-1 and DBF-2 to SPBs is constitutive and cell cycle independent. The three SIN kinases associate with SPBs of interphase nuclei as well as during early and late mitotic stages (as indicated by nuclear morphology). Nuclei were labeled with histone H1-RFP.
Figure 3.
CDC-7-dependent activation of DBF-2 occurs through SID-1 (A)
Reciprocal co-immunoprecipitation experiments of CDC-7-GFP and HA-SID-1 from cell extracts co-expressing both functionally tagged proteins indicate interaction of the two kinases. (B) In vitro DBF-2 activity assays revealed that addition of separately purified SID-1 stimulate DBF-2. SID-1-dependent stimulation of DBF-2 was further increased by addition of CDC-7 to the reaction. As control, CDC-7 alone did not stimulate DBF-2 (n = 5). To confirm equal precipitation of protein used for the kinase reactions, the kinase-sepharose pellet after the kinase reaction was boiled in Laemmli buffer and the supernatant used to determine equal protein abundance by SDS-PAGE and Western blot. (C) SID-1 was able to stimulate DBF-2, but not DBF-2(T671A). Western blot analysis of the precipitated proteins was used to determine comparable kinase levels (n = 5).
Figure 4.
Dual phosphorylation of DBF-2 is required for kinase activity and septum formation.
(A) Functional characterization of two conserved phosphorylation sites of DBF-2. The phosphomimetic DBF-2(T671E) variant complemented Δdbf-2, while substitution of Ser499 to alanine and glutamate and Thr671 to alanine did not. Cell wall and septa were labeled with Calcofluor White. (B) Kinase activity and MOB-1 interaction pattern of the indicated DBF-2 variants. Hydrophobic motif phosphorylation of Thr671 was required for maximal kinase activity, while either modification of Ser499 within the activation segment reduced DBF-2 activity to ca. 30% of the wild type DBF-2 control. Phospho-site double mutant analysis indicated that substitution of Thr671 to glutamate in a S499A and S499E background could only partly restore kinase activity. Precipitated DBF-2 variants were assayed in vitro using the synthetic NDR kinase peptide (KKRNRRLSVA) as substrate (n = 5). Western Blot analysis indicated equal precipitation of the co-activator protein MOB-1 with DBF-2 activation segment and hydrophobic motif variants.