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. 2013 Oct 15;126(20):4560–4571. doi: 10.1242/jcs.117663

Fig. 7.

Fig. 7.

Bem3 and secretory machinery proteins reciprocally regulate each other. (A) WT (BY4741) and bem3Δ cells expressing GFP-Sec4 (endogenous promoter) were grown overnight in selective medium at 30°C and imaged at 100× using a FITC filter. Fluorescence intensity of GFP–Sec4 was quantified using ImageJ and the polarized GFP–Sec4 fluorescence versus the bud/mother area ratio was plotted. *P<0.0001. (B) W303 WT cells expressing RFP-Sec4 (GAL1 promoter) and GFP-Bem3 were grown overnight in 2% glucose-containing medium, transferred to 2% galactose-containing medium for the indicated times and imaged at 100× using FITC and Rhodamine filters. Both Bem3 and Sec4 cluster in internal compartments of increasing size as Bem3 dosage increases. (C,D) W303 WT cells expressing GFP-Sec4 and Bem3PHm (C) or Bem3K1003A (D) were grown overnight in 2% glucose-containing medium, transferred to medium containing 2% galactose for 4 hours and then imaged at 100× using a FITC filter. (E) W303 WT cells expressing the indicated GFP-Sec4 mutants and Bem3 were grown overnight in 2% glucose-containing medium, transferred to medium containing 2% galactose for 4 hours and then imaged at 100× using a FITC filter. 24% of cells expressing GFP-Sec4Q79L showed the clustered phenotype upon Bem3 overexpression compared with 4% for GFP-Sec4S29N. (F,G) Cells expressing GFP-Bem3 and overexpressing Sec15 (F) or Sro7 (G) from a GAL1 promoter (pYES2.1) were grown in 2% galactose-containing medium for 15 hours before imaging at 100× using a FITC filter. Areas of the Bem3 compartments were measured as described above and plotted as a function of the bud/mother area ratio. Dashed lines indicate the boundaries of the corresponding bud/mother area ratios in WT cells grown under control conditions. (H) Working model of the relationship between the cell polarity protein Bem3 and vesicle trafficking. STEP I: Bem3 (red bars) recognizes sites of polarized growth through interactions with Cdc42 and PtdIns(4,5)P2, is retrieved from the plasma membrane by internalization and trafficked to a compartment reminiscent of the Spitzenkörper. STEP II: Bem3 actively recruits Sec4 (black asterisks) to this compartment. STEP III: Bem3 is recycled back to the bud tip from this Spitzenkörper-like compartment, in secretory vesicles in an Rcy1/Sec4-dependent manner. Scale bars: 5 µm.