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

Abstract

Our previous studies demonstrated that the key gluconeogenic enzyme fructose-1,6-bisphosphatase is secreted when Saccharomyces cerevisiae are starved of glucose for a prolonged period of time. In this study, we showed that malate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxykinase, glyceraldehyde-3-phosphate dehydrogenase, and cyclophilin A are also secreted in glucose-starved cells. Thus, both gluconeogenic and non-gluconeogenic enzymes are secreted via the non-classical pathway.

Keywords: :

• gluconeogenesis
• vacuole
• vacuole import and degradation
• non-classical secretion
• periplasm
• extracellular proteins
• fructose-1,6-bisphosphatase
• malate dehydrogenase
• phosphoenolpyruvate carboxykinase

When Saccharomyces cerevisiae are grown in media containing low glucose, gluconeogenic enzymes are induced.1-9 However, when glucose is added to prolonged-starved cells, these enzymes are inactivated and degraded in the vacuole via the Vid (vacuole import and degradation) pathway.10-24 These enzymes include fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase (MDH2), isocitrate lyase (Icl1p), and phosphoenolpyruvate carboxykinase (Pck1p).10-21,25,26 Recent evidence indicates that the key gluconeogenic enzyme FBPase is secreted when cells were starved of glucose for three days.22-24 Since other gluconeogenic enzymes MDH2, Icl1p, and Pck1p are also degraded in the vacuole via the same Vid pathway,16,25 they should show distribution patterns similar to those of FBPase. As such, they should also be secreted in glucose-starved cells. To test this, we used immuno-TEM techniques to determine the distribution of these enzymes at the ultra-structural level. To confirm our immune-TEM studies, we used an extraction procedure that extracts extracellular proteins from whole cells.22,24 We also determined whether or not non-gluconeogenic enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cyclophilin A (Cpr1p) were secreted in glucose-starved cells. GAPDH is a cell surface protein,27,28 whereas Cpr1p has been identified in the culture media in Saccharomyces cerevisiae in large-scale secretomic/extraction studies.29,30 Our results indicated that approximately 33.7–76.7% of these enzymes were secreted in glucose-starved cells. Given that these proteins do not contain typical ER signal sequence, they are secreted via the non-classical pathway. Thus, gluconeogenic as well as non-gluconeogenic enzymes utilize the un-conventional pathway to be secreted during prolonged starvation.

FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p are secreted in glucose-starved cells

We first used immuno-TEM to examine the distribution of FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p at the ultra-structural level. For localization studies, FBPase has previously been shown to be secreted into the extracellular space22,24 and was used as a positive control. Under the same experimental conditions, the coatomer subunit Sec28p is distributed mainly in the intracellular fraction as shown previously22,24 and was used as a negative control. Because antibodies against Pck1p and Icl1p were not available, we produced a wild-type strain expressing Icl1p tagged with HA and Pck1p-tagged with Myc. We then used HA and Myc antibodies to detect these tagged proteins. For these experiments, wild-type cells co-expressing Icl1p-HA and Pck1p-Myc were grown in YPKG media containing low glucose for three days and processed for immuno-TEM studies. First, we used western blotting to examine whether or not these antibodies recognized proteins at the expected molecular weights in total lysates. As is shown in Figure 1A, polyclonal antibodies against Sec28p, FBPase, MDH2, HA, Myc, GAPDH, and Cpr1p all reacted with a single protein band at the predicted molecular weights.

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.

We next examined the distribution of these proteins by incubating thin sections of glucose-starved wild-type cells in the absence or presence of purified primary antibodies followed by secondary antibodies conjugated with 10 nm gold particles (Fig. 1B). When thin sections of cells were incubated in the absence of primary antibodies, there were very few gold particles observed between the cell wall and the plasma membrane. In a similar manner, low levels of Sec28p were detected in the extracellular space. In contrast, substantial amounts of the positive control protein FBPase were present in areas between the cell wall and the plasma membrane. Likewise, significant amounts of MDH2, Icl1p-HA, Pck1p-Myc, GAPDH, and Cpr1p were also found in the extracellular space in cells that were grown in low glucose for a prolonged period of time. Quantification of immunogold particles indicated that approximately 76.7% of FBPase, 36.9% of MDH2, 46.9% of Icl1p, 57.6% of Pck1p, 33.7% of GAPDH, and 42.7% of Cpr1p were in the extracellular space in glucose-starved cells (Table 1).

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.

We next used the extraction procedure to confirm the presence of these proteins in the extracellular fraction.22,24 Wild-type cells expressing Icl1p-HA and Pck1p-Myc were grown in low-glucose media for three days and subjected to the extraction procedure. Following the extraction protocol, proteins were separated into the intracellular and extracellular fractions. The distribution of these proteins in these fractions was then examined by western blotting (Fig. 2). Scw4p is the soluble cell wall protein31 and was used as a control for proteins that reside in the extracellular fraction. Not surprisingly, the majority of Scw4p was in the extracellular fraction. In contrast, most of the Sec28p was distributed in the intracellular fraction. As expected, significant amounts of FBPase were in the extracellular fraction. In a similar manner, MDH2, Icl1p-HA, Pck1p-Myc, GAPDH, and Cpr1p were all found in the extracellular fraction. Taken together, our results indicate that FBPase, MDH2, Icl1p-HA, Pck1p-Myc, GAPDH, and Cpr1p are secreted when cells were grown in media containing low glucose. Because these enzymes do not contain typical ER signal sequence, they are secreted by the non-conventional pathway.

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.

Secretion of signal-less proteins in extracellular vesicles has been observed in different fungi including Cryptococcus neoformans, Candida parapsilosis, Candida albicans, Histoplasma capsulatum, Paracoccidiodes brasiliensis, Sporothrix schenckii, Saccharomyces cerevisiae, and Malassezia sympodialis.30,32-40 Extracellular vesicles isolated from different species showed differences in their sizes. For example, 20–400 nm vesicles were observed in C. neoformans, while 10–350 nm vesicles and 20–200 nm vesicles were found in H. capsulatum and in P. brasiliensis respectively.36-38 Extracellular vesicles of 50- 250 nm were also detected in culture media from S. cerevisiae.36 Interestingly, FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p were identified in extracellular vesicles isolated from Histoplasma capsulatum.36 We have shown previously that FBPase is associated with small vesicles (Vid vesicles) in the cytoplasm in S. cerevisiae.26 These vesicles exist in at least two forms. Free vesicles are 30–50 nm in diameter.26 Vid vesicles can also aggregate to form large clusters in the cytoplasm.15 Given that substantial amounts of gluconeogenic enzymes are secreted in glucose-starved cells, these proteins may also be secreted in vesicles. Future experiments will be needed to elucidate the molecular mechanisms responsible for the secretion of signal-less proteins and vesicles across the plasma membrane and the cell wall.

Methods

Cell Culture, Media and Antibodies

Yeast strains used in this study included wild-type (BY4742, MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0), wild-type tagged with Icl1p-HA and Pck1p-Myc (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 trp1–1 Icl1p-HA-TRP1 Pck1p-Myc-HIS3), and wild-type expressing Scw4p-GFP (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 Scw4p-GFP::HIS3). Cells were first grown in YPKG (1% yeast extract, 2% peptone, 1% potassium acetate and 0.5% glucose) for three days and processed. Antigen affinity purified anti-GAPDH polyclonal antibodies were purchased from Protein Tech Group. Affinity purified anti-HA polyclonal antibodies were purchased from Immunology Consultant Laboratory. Affinity purified polyclonal anti-Myc antibodies were obtained from Santa Cruz Biotechnology. Wild-type cells expressing Scw4p-GFP were purchased from Invitrogen. Anti-GFP polyclonal antibodies were purchased from Abcam.

Immuno-TEM

Immuno-TEM was performed as described.22 After three days in YPKG media, cells were fixed with 3% paraformaldehyde and 0.2% glutaraldehyde overnight at room temperature. Cells were serially dehydrated and embedded in LR White. Embedded samples were sectioned and placed onto Formvar-carbon-coated nickel grids. Grids containing cell sections were incubated on a droplet containing 0.1% BSA/PBS for 30 min. Next, the grids were incubated with or without primary antibody (Sec28p:10 x, FBPase: 50 x, MDH2: 20 x, pHA: 20 x, pMyc: 20 x, GAPDH: 10 x, and Cpr1p: 20 x) diluted in 1% BSA/PBS for 2 h. Grids were then washed and incubated in 50 x dilution of goat anti-rabbit IgG conjugated with 10 nm immunogold particles (Ted Pella). After washing, grids were stained with 2% uranyl acetate for 1 min and viewed on a JEOL JEM-1400 electron microscope with an Orius SC1000 CCD camera.

Extraction and Western Blotting

Extraction was performed as described.22,24 Cells were grown in 2 ml YPKG for three days and harvested. Cells (OD₆₀₀ = 10/ml) were incubated with 100 μl of extraction buffer containing 0.1 M Tris, pH 9.4 and 10 mM β-mercaptoethanol in a 37°c shaker for 15 min. Following incubation, cells were pelleted at 3,000 x g for 5 min. The supernatant fraction was precipitated with 15% TCA, washed and resuspended in SDS-PAGE buffer. The distribution of proteins in the extracellular and intracellular fractions was examined using polyclonal antibodies directed against Sec28p, FBPase, MDH2, HA, Myc, GAPDH, and Cpr1p followed by peroxidase-conjugated donkey anti-rabbit IgG diluted 1:10,000 (GE Healthcare). The distribution of Scw4p-GFP was examined using anti-GFP antibodies. Bands were developed using Western Lighting Plus ECL (Perkin Elmer).

Abbreviations:
fructose-1,6-bisphosphatase	=	FBPase
malate dehydrogenase	=	MDH2
isocitrate lyase	=	Icl1p
phosphoenolpyruvate carboxykinase	=	Pck1p
glyceraldehyde-3-phosphate dehydrogenase	=	GAPDH
cyclophilin A	=	Cpr1p
vacuole import and degradation	=	Vid

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Immuno-TEM was performed using the EM Core Facility at Penn State College of Medicine. This work was supported by Penn State Bridge Fund to Hui-Ling Chiang.