Multistep and multimode cortical anchoring of tea1p at cell tips in fission yeast

Abstract

The fission yeast cell-polarity regulator tea1p is targeted to cell tips by association with growing microtubule ends. Tea1p is subsequently anchored at the cell cortex at cell tips via an unknown mechanism that requires both the tea1p carboxy-terminus and the membrane protein mod5p. Here, we show that a tea1p-related protein, tea3p, binds independently to both mod5p and tea1p, and that tea1p and mod5p can also interact directly, independent of tea3p. Despite their related structures, different regions of tea1p and tea3p are required for their respective interactions with an essential central region of mod5p. We demonstrate that tea3p is required for proper cortical localization of tea1p, specifically at nongrowing cell tips, and that tea1p and mod5p are independently required for tea3p localization. Further, we find that tea3p fused to GFP or mCherry is cotransported with tea1p by microtubules to cell tips, but this occurs only in the absence of mod5p. These results suggest that independent protein-protein interactions among tea1p, tea3p and mod5p collectively contribute to tea1p anchoring at cell tips via a multistep and multimode mechanism.

Figure 1

Tea1p, mod5p and tea3p form independent complexes in vivo. (A) GST-mod5p was immunoprecipitated from soluble protein extracts of wild-type cells expressing GST-mod5p and tea3p-GFP (lanes 1 and 5), mod5Δ cells expressing tea3p-GFP (lanes 2 and 6), tea3Δ cells expressing GST-mod5p (lanes 3 and 7) and tea1Δ cells expressing GST-mod5p and tea3p-GFP (lanes 4 and 8). The resulting immunocomplexes were analyzed for GST-mod5p, tea3p-GFP and tea1p. Whole-cell extract (WCE) fractions are shown in lanes 1–4 and immunoprecipitates in lanes 5–8. Immunoprecipitates were loaded 30 × relative to WCE sample. (B) Tea3p-HA was immunoprecipitated from soluble protein extracts of wild-type cells (lanes 1 and 4), wild-type cells expressing tea3p-HA (lanes 2 and 5) and mod5Δ cells expressing tea3p-HA (lanes 3 and 6). The resulting immunocomplexes were analyzed for tea3p-HA and tea1p. WCE fractions are shown in lanes 1–3 and immunoprecipitates in lanes 4–6. Immunoprecipitates were loaded 20 × relative to WCE. (C) Schematic diagram summarizing interactions between tea1p, tea3p and mod5p. (D) GST-mod5p (lanes 3 and 4) or tea1p (lanes 5 and 6) was immunoprecipitated from soluble protein extracts of either wild-type (lanes 1, 3 and 5) or tea1Δ200 (lanes 2, 4 and 6) cells expressing GST-mod5p and tea3p-HA. The resulting immunocomplexes were analyzed for GST-mod5p, tea1p (or tea1Δ200p) and tea3p-HA. WCE fractions are shown in lanes 1 and 2 and immunoprecipitates in lanes 3–6. Immunoprecipitates were loaded 30 × relative to WCE.

Figure 2

Mod5p amino acids 156–255 are essential for the localization of tea1p and mod5p. The localization of tea1p (green) and microtubules (red) in (A–D) mod5Δ cells and (I–L) wild-type cells, expressing different mutant versions of GFP-mod5p. (E–H) The localization of GFP-mod5p (and mutant versions) in mod5Δ cells. (A, E, I) Wild-type GFP-mod5p; (B, F, J) GFP-mod5Δ256–305p; (C, G, K) GFP-mod5Δ156–205p; (D, H, L) GFP-mod5Δ206–255p. The scale bar represents 5 μm.

Figure 3

Localization dependencies of tea3p-GFP. Localization of tea3p-GFP in (A) wild-type, (B) tea1Δ, (C) tea1Δ200, (D) mod5Δ, (E) mod5Δ tea1Δ and (F) mod5Δ206–255 cells. The scale bar represents 5 μm.

Figure 4

Microtubule-dependent movement of tea3p in mod5Δ. (A) Time-lapse movie frames of tea3p-GFP in mod5Δ cells at 15 s intervals. Red and turquoise arrowheads mark traveling particles of tea3p-GFP. (B) Time-lapse movie frames of GFP-atb2p (green) and tea3p-mCh (red) in mod5Δ cells at 15 s intervals. Gray dashed line indicates the starting position of the traveling tea3p-mCh particle. (C) Localization of GFP-atb2p (green) with tea3p-mCh (red) in wild-type cells. (D) Localization of tea1p-GFP (green) with tea3p-mCh (red) in wild-type cells. (E, F) Time-lapse movie frames of tea1p-GFP (green) and tea3-mCh (red) in mod5Δ cells at 15 intervals. Traveling particles of colocalized tea1p-GFP and tea3p-mCh are indicated by white arrowheads and static particles are indicated by white arrows. (G) Time-lapse movie frames of tea3p-GFP in mod5 tea1Δ cells at 15 s intervals. Red dotted lines indicate reduced movement of tea3p-GFP. The scale bar represents 5 μm.

Figure 5

Mod5p and tea3p have distinct but overlapping functions. (A) Wild-type, mod5Δ, tea3Δ, tea1Δ, mod5Δ tea3Δ, mod5Δ tea1Δ, tea3Δ tea1Δ and tea1Δ200 cells were depolarized by growth to stationary phase and returned to fresh medium in the absence (gray bars) or presence (black bars) of 50 μg/ml MBC for 3 h at 32°C. The percentage of branched cells in each sample was counted, n=200. (B) Percentage of daughter cell pairs displaying illustrated initial growth patterns after septation in wild-type (n=133), mod5Δ (n=228), tea3Δ (n=165) and tea1Δ cells (n=194). Arrows indicate direction of growth.

Figure 6

Tea1p is preferentially lost from the nongrowing tip in tea3Δ cells. (A–H) Treatment of (A, D) wild-type, (B, E) tea3Δ and (C, F) orb2-34 cells with MBC for (A–C) 0 min or (D–F) 5 min. Cells are stained for tea1p (green) and microtubules (red). Time course of tea1p loss from cell tips in (G) wild-type and (H) tea3Δ cells, showing number of cells with detectable levels of tea1p at one cell tip, two tips or neither tip, for each strain. n=200 for each strain. (I–N) Treatment of tea3Δ cells with MBC for (I–K) 0 min or (L–N) 5 min. Cells are stained with anti-tea1p antibodies (I, L) and Alexa-labeled phalloidin (J, M). Merged imaged are shown with tea1p in green and phalloidin in red (K, N). The scale bars represent 5 μm.

Figure 7

Multistep cortical retention of tea1p. A model of protein–protein interactions regulating tea1p localization at cell tips. (A) Tea1p arrives at the cell cortex on microtubule plus ends. (B) The tea1p N-terminus interacts with mod5p at both cell tips. (C) At nongrowing cell tips, the tea1p C-terminus interacts with tea3p, which is itself anchored at the cortex (D) via interaction with mod5p. (E) At growing cell tips, tea1p interacts with unknown factors (X) that provide a function similar to that of tea3p at nongrowing tips. (F) A partially functional interaction between tea1p and tea3p can occur without mod5p, but tea1p and tea3p will be poorly anchored. (G) Together, these interactions lead to proper tea1p anchoring at both cell tips. See text for further details.