Fructose-1,6-bisphosphatase, Malate Dehydrogenase, Isocitrate Lyase, Phosphoenolpyruvate Carboxykinase, Glyceraldehyde-3-phosphate Dehydrogenase, and Cyclophilin A are secreted in Saccharomyces cerevisiae grown in low glucose

Figures & data

Figure 1. Immuno-TEM analysis of the distribution of FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p in glucose-starved wild-type cells. (A), wild-type cells expressing Icl1p-HA and Pck1p-Myc were lysed and total lysates were examined by western blotting using antibodies directed against Sec28p, FBPase, MDH2, HA, Myc, GAPDH, and Cpr1p. (B), wild-type cells expressing Icl1p-HA and Pck1p-Myc were starved of glucose for 3 d and fixed. Cells were processed and embedded. Thin sections (10 nm) were incubated in the absence or presence of primary antibodies directed against Sec28p, FBPase, MDH2, HA, Myc, GAPDH, and Cpr1p followed by secondary antibodies conjugated with 10 nm gold particles. Enlargements of the periplasm in sections of the whole cells were shown. CW:cell wall. PM:plasma membrane. Bars: 200 nm.

Table 1. Quantification of the number of gold particles per cell in the I and E fractions

		Intracellular		Extracellular
No primary antibodies		4.1 ± 2.1		3.2 ± 1.3
Sec28p		60.3 ± 10.2		3.5 ± 2.3
FBPase		49.7 ± 13.1		165.8 ± 23.5
MDH2		121.1 ± 15.3		70.6 ± 9.9
Icl1p-HA		88.3 ± 10.1		78.2 ± 5.9
Pck1p-Myc		130.8 ± 18.3		178.3 ± 21.2
GAPDH		117.9 ± 33.9		59.8 ± 15.2
Cpr1p		94.5 ± 1.9		70.3 ± 14.2

Thin sections of wild-type cells expressing Icl1p-HA and Pck1p-Myc were incubated in the absence or presence of primary antibodies directed against Sec28p, FBPase, MDH2p, HA, Myc, GAPDH, and Cpr1p followed by secondary antibodies conjugated with 10 nm gold particles. The number of gold particles per cell in the intracellular (I) and extracellular/periplasmic fraction (E) was counted. Mean and Standard Error of the Mean (SEM) were obtained by counting the number of gold particles in the intracellular and extracellular fractions from 3 micrographs.

Figure 2. . FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p are in the extracellular fraction in glucose-starved cells. Wild-type cells expressing Scw4p-GFP and wild-type cells co-expressing Icl1p-HA and Pck1p-Myc were grown in YPKG for three days and subjected to the extraction protocol. The distribution of Scw4p-GFP in the intracellular and extracellular fractions was examined by anti-GFP antibodies. The distribution of Sec28p, FBPase, MDH2, Icl1p-HA, Pck1p-Myc, GAPDH, and Cpr1p in the intracellular and extracellular fractions was examined using western blotting.