Figure 1.
Summary of phosphoproteomics results conducted on Δmak-2Q100G mutant.
(A) Overview of FunCat categories of proteins harboring the identified phosphopeptides. The upper bar shows the categories of all the proteins with identified phosphopeptides (3200 phosphopeptides, 1164 proteins), the middle bar shows functional categories of proteins with phosphopeptides that showed higher abundance after inhibition (33 phosphopeptides, 27 proteins) and the lower bar shows FunCat analysis of the proteins with phosphopeptides that showed lower abundance in mak-2Q100G germlings after treatment with 1NM-PP1 (96 phosphopeptides, 67 proteins). (B) Pie chart showing the relative percentages and absolute numbers of single, double, triple and quadruple phosphosites per peptide. (C) Pie chart showing the relative percentages and absolute numbers of phosphorylated serine, threonine and tyrosine in all peptides found.
Table 1.
Phosphopeptides with predicted MAPK site that showed decreased abundance in 1NM-PP1-treated mak-2Q100G cells.
Figure 2.
HAM-5-GFP localization in WT and Δmak-2 germlings.
(A) Schematic overview of HAM-5 protein structure. The predicted WD40 domains are shown in grey and the putative coiled coil domains are shown as red bars. The two disordered regions with low complexity are depicted by shaded white boxes. The MAPK phosphorylation site (aa 506) is marked by a blue star, the other two sites showing decreased abundance in treated cells (aa 1288 and 1604) are marked by green stars, and other 13 identified phosphorylation sites (S14, S414, S792, S818, S833, T838, T969, S1085, S1199, T1201, S1202, T1353, S1608) [40] are marked by black stars. The putative MAPK docking site is marked by a yellow line. (B) Localization of HAM-5-GFP to puncta localized to CAT tips during chemotropic interactions between genetically identical cells. HAM-5-GFP showed dynamic localization to CAT tips of germlings with an oscillation of every four min (arrow). HAM-5-GFP also localized to puncta within germlings and near nuclear compartments devoid of HAM-5-GFP (asterisks). The image left is a bright field image. Scale bar = 10 µM. (C) HAM-5-GFP localized to the sites of contact during germling fusion (arrow). (D) Western blots of WT, WT (ham-5-gfp) and Δmak-2 (ham-5-gfp) germlings with immunoprecipitated HAM-5-GFP probed with anti-GFP antibodies (right panel shows longer run showing higher mobility of HAM-5-GFP in wild type germlings) specifically detecting HAM-5-GFP (210 kD; Figure S4D). Lower panel shows a Western blot with identical samples probed with anti-phospho antibodies that specifically detect phosphorylated serine or threonine residues followed by a proline. (E) Localization of HAM-5-GFP to puncta in Δmak-2 germlings. Some puncta showed localization to germling tips, but which did not oscillate during growth (white arrows). Scale bar = 10 µM.
Figure 3.
HAM-5-GFP shows localization with components of the MAK-2 pathway.
(A) Co-localization of HAM-5-GFP and MAK-2-mCherry during germling communication (arrows). (B) Co-localization of HAM-5-GFP and MEK-2-mCherry during germling communication (arrows). (C) Co-localization of HAM-5-GFP and NRC-1-mCherry during germling communication. NRC-1-mCherry strains show low fluorescence [18]. (D) HAM-5-GFP and SO-mCherry do not co-localize during chemotropic interactions, but instead show opposite localization to CAT tips in communicating germlings (arrows). The images on the left are bright field images, fluorescent images on the right. Scale bar = 10 µM. (E) Co-immunoprecipitation experiments showing an interaction between HAM-5-GFP (210 kD; Figure S4D) and MEK-2-mCherry (82.9 kD; Figure S4C) and NRC-1-mCherry (128 kD; Figure S4C). Input panels show Western blots of immunoprecipitated protein samples from 5 hr-old germlings probed with either anti-GFP (free GFP = 27 kD; Figure S4D) or anti-mCherry antibodies. The output panel is a Western blot of proteins immunoprecipitated by anti-GFP antibodies (and thus HAM-5-GFP) and probed with anti-mCherry antibodies (detecting MEK-2-mCherry or NRC-1-mCherry).
Figure 4.
The WD40 domain of HAM-5 interacts with MAK-2, while the C-terminus interacts with MEK-2.
(A) Localization of HAM-51-351-GFP (WD40 domain only) in WT germlings localized to the cytoplasm, the nucleus and the puncta at the cell periphery and at the tip during chemotropic interactions and oscillation (arrow). (B) HAM-51-351-GFP in Δham-5 germlings localized to the cytoplasm and the nucleus; no puncta were observed. The images on the left are bright field images. Scale bar = 10 µM. (C) Western blots showing a specific interaction between MEK-2-mCherry (82.9 kD) with full length HAM-5-GFP (210 kD) or HAM-5Δ67-348-GFP (180 kD), but not with free GFP (27 kD; Figure S4C, D). An interaction between MEK-2-mCherry and HAM-51-351-GFP (65.3 kD) was not detected. Input panels show Western blot of immunoprecipitated proteins isolated from 5 hr-old germlings probed with either anti-mCherry antibodies or anti-GFP antibodies. Output panel shows anti-mCherry immunoprecipitated proteins (MEK-2-mCherry) probed with anti-GFP antibodies (HAM-5-GFP). (D) Western blots showing a specific interaction between full length HAM-5-GFP or HAM-51-351-GFP and phosphorylated MAK-2. Input panels show Western blot of proteins isolated from 5 hr-old germlings probed with anti-p42/44 antibodies (which recognize phosphorylated MAK-2 (40.8 kD; Figure S4D, E) [8]) or immunoprecipitated proteins probed with anti-GFP antibodies. The output panel shows anti-GFP immunoprecipitated protein sample probed with anti-p42/44 antibodies, showing interaction between HAM-5-GFP or HAM-51-351-GFP and phosphorylated MAK-2. (E) Schematic showing regions of interaction between HAM-5 and MAK-2 or MEK-2 based on co-immunoprecipitation experiments.
Figure 5.
HAM-5 is required to localize MAK-2 and MEK-2 to puncta.
(A) MAK-2-GFP in isolated WT germlings localizes to the nucleus, cytoplasm and to puncta (white arrows). (B) In the Δham-5 mutant, MAK-2-GFP localization is cytoplasmic and nuclear; no puncta are observed. Scale bar = 10 µM. (C) In WT hyphae, MEK-2-mCherry localizes to the septum (green arrow) and also to cytoplasmic puncta (white arrow). Scale bar = 10 µM. (D) In Δham-5 hyphae, septum localization of MEK-2-mCherry is observed (green arrow), but puncta are not. Scale bar = 10 µM. (E) Co-immunoprecipitation experiments showing an interaction between MEK-2-mCherry and phosphorylated MAK-2 in WT (mek-2-mCherry) germlings, but a significant reduction in interaction between MAK-2 and MEK-2-mCherry in a Δham-5 (mek-2-mCherry) strain. Top panel is a Western blot of protein samples probed with anti-p42/44 antibodies (MAK-2, 40.8 kD, MAK-1, 46.7 kD; Figure S4E). Middle panel is anti-mCherry immunoprecipitated proteins probed with anti-mCherry antibodies (MEK-2-mCherry, 82.9 kD; Figure S4C, E). Bottom panel is anti-mCherry (MEK-2-mCherry) immunoprecipitated proteins probed via Western blot with anti-p42/44 antibodies that recognize phosphorylated MAK-2.
Figure 6.
HAM-5-GFP and MAK-2-mCherry localize to puncta in Δham-7 and Δham-11 fusion-deficient germlings.
(A) Western blot of protein samples from 5 hr-old WT, Δham-5, Δham-7 and Δham-11 germlings probed with anti-p42/44 antibodies, which recognize phosphorylated MAK-1 and MAK-2 [8]. As previously shown [43], MAK-1 phosphorylation is reduced in the Δham-7 mutant. (B) HAM-5-GFP and MAK-2-mCherry localization in WT germlings undergoing chemotropic interactions. Arrows show localization to the CAT tip and to cytoplasmic puncta. (C) HAM-5-GFP and MAK-2-mCherry co-localization in Δham-7 (ham-5-gfp; mak-2-mCherry) germlings. Note lack of chemotropic interactions. Arrows show puncta of HAM-5-GFP and MAK-2-mCherry in Δham-7 (ham-5-gfp; mak-2-mCherry) germlings, which are often co-localized at the germ tube tip. (D) HAM-5-GFP and MAK-2-mCherry co-localization in Δham-11 (ham-5-gfp; mak-2-mCherry) germlings. Note lack of chemotropic interactions. HAM-5-GFP and MAK-2-mCherry co-localized to both cytoplasmic and tip-localized puncta (arrows). The upper left panels are bright field images, the upper right panels show GFP fluorescence, the lower left panels show mCherry fluorescence. Lower right panels show merged images of GFP and mCherry images. Scale bars = 10 µM.
Figure 7.
HAM-5-GFP shows oscillatory localization to fusion points and puncta in hyphae showing chemotropism.
(A) Time course of HAM-5-GFP localization to interacting hyphae prior to cell fusion. HAM-5-GFP localized to the hyphal tip of a homing hyphae (white arrow T = 0; T = 8), followed by a disappearance and localization of HAM-5-GFP at the cell surface in the receptive hypha (white arrow; T = 4). Red arrow shows localization to septa near fusion points. At T = 30, HAM-5 is observed at the site of contact (white arrow). Bright field image is shown in upper left panel; remaining panels show GFP fluorescence. Scale bar = 50 µM. (B) Graphical representation of relative fluorescence intensity (R.F.I.) of HAM-5-GFP localization to the receptive hypha and the homing hypha over the time course (panel A). × axis shows time (min). (C) Graphical representation of HAM-5-GFP fluorescence of interacting fusion hyphae shown in Figure S6B over an extended time course. Note that following the fusion event (T = 22 min), HAM-5-GFP puncta co-oscillate in both hyphae for an additional 30 minutes, see (D). y axis shows maximal fluorescence intensity (M.F.I.) while the × axis shows time (min). The time points at which the individual pictures taken at T = 4, T = 8, T = 46 and T = 59 minutes are pointed out in the graph (black arrows) (Figure S6 and Movie S5). The time of fusion at T = 22 min is marked with a black line. (D) Example of HAM-5-GFP appearing in puncta in both hyphae after fusion at T = 46.
Figure 8.
Model for HAM-5-MAK-2/MEK-2/NRC-1 function during chemotropic interactions.
HAM-5 interacts with MAK-2, MEK-2 and NRC-1 and assembles in puncta throughout the germling, some of which are recruited to the CAT tip during chemotropic interactions via interactions with a plasma membrane associated protein (MDP: membrane docking protein). Association of the HAM-5/MAK-2/MEK-2/NRC-1 complex to the CAT tips is associated with signal reception from the partner germling. During this process, HAM-5 is successively phosphorylated by MAK-2 and other kinases, resulting in the disassociation of the complex and termination of the ability to receive signal. Nuclear MAK-2 signaling to the transcription factor PP-1 is not believed to be essential for chemotropic interactions as treatment of germlings with cycloheximide did not disturb oscillation of MAK-2 nor chemotropic behavior [19]. The coordinated assembly and disassembly of HAM-5/MAK-2/MEK-2/NRC-1 during communication regulates the tempo of chemotropic interactions between germlings.