Table 1.
Lists of DNA primers used in this study.
Fig 1.
Physical maps of plasmids harboring mPmRab7 and pVP28 fused with half 3’-agglutinin of S. cerevisiae for surface display by P. pastoris.
Fig 2.
Immunofluorescence microscopy of mPmRab7-Agα1p and pVP28-Agα1p surface displayed by P. pastoris.
Methanol induced His+MutS transformants with (A) mPmRab7-Agα1p, and (B) pVP28-Agα1p grown at 20°C for 96 h and incubated with polyclonal rabbit anti-PmRab7 and anti-VP28, respectively. Panel (a) is light microscope, (b) and (c) are fluorescent microscope where (c) is the enlargement of some group of cells in (b). Fluorescence was observed on the cell surface more than in (C) the Agα1p control. Bar = 5 μm, fluorescence magnification = 1,800 ×.
Fig 3.
Quantification of mPmRab7 and pVP28 on the cell surface of P. pastoris by (A) indirect ELISA and (B) sandwich ELISA.
The quantity of methanol-induced His+MutS yeast cells was 108 cells/ml (OD600 = 10). The values were obtained from three independent experiments. Bar = means ± SD.
Fig 4.
Yeast agglutination and agglutination inhibition assays.
(A) OD600 of His+MutS yeast cells induced by methanol for 24–96 h, mixed at initial OD600 = 0.7 and measured after 1 h. Percent agglutination was calculated from OD600 at time zero and after 1 h. (B) Percent inhibition of agglutination by treatment of yeast cells with PmRab7 and VP28 antibodies for 1 h prior to agglutination assay. Percent inhibition was calculated as percent of agglutination with and without specific antibody treatment. The values were obtained from three independent experiments. Bar = means ± SD (Some of the SD bars are so small that they cannot be seen in the graph).
Fig 5.
(A) Results for negative control Agα1p only and showing approximately 20% autofluorescence. (B) Yeast cells expressing mPmRab7-Agα1p and showing fluorescence for approximately 90% of total events. (C) Yeast cells expressing pVP28-Agα1p and showing fluorescence for approximately 70% of total events. The data was selected by relative statistical value from three independent experiments.