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Cell Biology International (2008) 32, 502–504 (Printed in Great Britain)
G-protein-coupled receptor Gpr1 and G-protein Gpa2 of cAMP-dependent signaling pathway are involved in glucose-induced pexophagy in the yeast Saccharomyces cerevisiae
Volodymyr Y. Nazarkoa, Johan M. Theveleinbc and Andriy A. Sibirnyad*
aInstitute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005, Ukraine
bDepartment of Biology, Katholieke Universiteit Leuven, Belgium
cDepartment of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium
dDepartment of Biotechnology and Microbiology, Rzeszów University, Ćwiklińskiej 2, 35-601 Rzeszów, Poland


Abstract

In yeast cell, glucose induces various changes of cellular metabolism on genetic and metabolic levels. One of such changes is autophagic degradation of dispensable peroxisomes (pexophagy) which occurs in vacuoles. We have found that in Saccharomyces cerevisiae, defect of G-protein-coupled receptor Gpr1 and G-protein Gpa2, both the components of cAMP-signaling pathway, strongly suppressed glucose-induced degradation of matrix peroxisomal protein thiolase. We conclude that proteins Gpr1 and Gpa2 are involved in glucose sensing and signal transduction during pexophagy process in yeast.


Keywords: cAMP signalling pathway, Pexophagy, Gpr1, Gpa2, Yeast, Saccharomyces cerevisiae.

*Corresponding author. Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005, Ukraine.


1 Introduction

Eukaryotic cells adapt to environmental changes using numerous strategies, including induction and repression of synthesis of some proteins as well as degradation of another ones. Not only redundant proteins but also non-essential at the moment organelles (peroxisomes, mitochondria) can be effectively degraded if demand of cell in them has been decreased. Organelles and many proteins are ultimately delivered to vacuole (lysosome) and are degraded in so named autophagy pathway (Klionsky and Ohsumi, 1999). In the case of peroxisome autophagic degradation, this process is known as pexophagy. Labeling and delivery of redundant proteins and organelles to vacuole for degradation are rather complex process which is controlled at least by 30 specific genes known as ATG (Klionsky et al., 2003; Dunn et al., 2005).

In yeasts, peroxisome propagation can be induced by specific carbon sources, such as oleic acid or methanol known as peroxisome proliferators. Peroxisomes contain enzymes involved in major steps of catabolism of peroxisome proliferators. However, these compounds are less favorable substrates relative to glucose which is known to induce pexophagy (Dunn et al., 2005). An other carbon source known to specifically induce pexophagy (only in methylotrophic yeasts) is ethanol (Kulachkovsky et al., 1997). How cell senses glucose and transduces glucose signal to cause all subsequent steps of intracellular cascade events leading to pexophagy is not known. In the methylotrophic yeast Hansenula polymorpha, the glucose sensor Gcr1 was identified recently (Stasyk et al., 2004). Point mutation and knock out of GCR1 gene led to practically total defect in glucose catabolite repression involved in synthesis of peroxisomal and cytosolic enzymes whereas glucose-induced pexophagy was only slightly affected. It was suggested that glucose sensing for pexophagy is differed from that for catabolite repression (Stasyk et al., 2004). It is known that in Saccharomyces cerevisiae glucose sensing for catabolite repression, hexose transporter induction, and activation of cAMP-signaling pathway involved in trehalose and glycogen mobilization and in loss of stress resistance are occurred by different proteins (Kraakman et al., 1999; Thevelein et al., 2005; Bellinchon and Gancedo, 2007).

In general, early steps of both specific pexophagy and general autophagy are poorly understood. It was found that both cAMP and Tor signaling pathways are involved in early stages of general autophagy induced by nitrogen starvation or by antibiotic rapamycin (Stromhaug and Klionsky, 2001; Budovskaya et al., 2004), however, nothing is known on sensing mechanism of this process.

We have suggested that G-protein-coupled receptor Gpr1 and interacting with it G-protein Gpa2 are both involved in glucose sensing/signaling during pexophagy. Data presented in this communication clearly show that defects in sensing components of cAMP-signaling pathway due to mutations in GPR1 and/or GPA2 genes lead to strong defect in glucose-induced degradation of peroxisomal thiolase in S. cerevisiae. We suggest that Gpr1 and Gpa2 proteins are main components in glucose sensing during pexophagy process in yeast.

2 Materials and methods

2.1 Strains and media

S. cerevisiae strains used in this study are listed in Table 1.


Table 1.

S. cerevisiae strains used in study

StrainRelevant genotypeReference
W303-1AMATa leu2-3112 ura3-1 trp1-92 his3-11,15 ade2-1 can1-100 GAL SUC malKraakman et al. (1999)
PM731W303-1A gpa2two colonsURA3Kraakman et al. (1999)
LK5W303-1A gpr1two colonsLEU2Kraakman et al. (1999)
LK6W303-1A gpr1two colonsLEU2 gpa2two colonsURA3Kraakman et al. (1999)


Yeast strains were pregrown in YPD (1% yeast extract, 1% peptone, and 1% glucose). Peroxisomes were induced during growth in oleic acid minimal medium (YOA: 0.67% yeast nitrogen base, 0.1% Tween 40 and 0.1% oleic acid and appropriate amino acids, uracil, adenine, as required). For initiation of pexophagy, glucose minimal medium without nitrogen source YG(–N) medium (0.17% yeast nitrogen base without ammonium sulfate, 2% glucose and appropriate amino acids, uracil, adenine, as required) was used.

2.2 Pexophagy assays

For pexophagy assay the yeast cells were grown overnight in 3ml of liquid YPD medium. The next day 1ml of the overnight culture was transferred into 20ml of fresh YPD medium and pregrown for 6h. Then cells were washed twice with distilled water and transferred into 20ml of YOA medium with starting OD600=0.3 for induction of peroxisome biogenesis for 23h. Induced cells were pelleted, resuspended in 0.5ml of distilled water and transferred into 20ml of YG(-N) medium with starting OD600=0.25. An equal volume of liquid culture of each strain was withdrawn at 0, 10, 15 and 20h of glucose adaptation for preparing protein samples which subsequently were used for immunoblot analysis, as described previously (Baerends et al., 2000). Western blotting was made using rabbit antibodies against S. cerevisiae peroxisomal matrix protein thiolase, as described before (Cao and Klionsky, 2007).

3 Results

During growth of the yeast S. cerevisiae in minimal medium with oleic acid as sole carbon and energy source, peroxisome biogenesis is induced (Hutchins et al., 1999). When the culture is shifted to glucose minimal medium without nitrogen source, the yeast cells adapt to novel environmental conditions by degrading not longer needed peroxisomes in a process known as autophagic peroxisome degradation or pexophagy. To test, if glucose sensing in specific autophagic peroxisome degradation (pexophagy) occurs through cAMP-dependent signaling pathway, we studied kinetics of degradation of peroxisomal matrix protein, thiolase Fox3, during adaptation of oleate-grown cells to glucose minimal medium without nitrogen source. Cells of the wild-type strain as well as mutants gpr1 (defective in G-protein-coupled receptor, GPCR), gpa2 (defective in interacting with GPCR G-protein) and double gpr1 gpa2 mutant were used in this experiment. After shift to glucose medium, the amount of thiolase protein was decreased in the wild-type cells whereas this process was strongly retarded in single gpr1 and gpa2 as well as double gpr1 gpa2 mutants (Fig. 1). So, these data unequivocally show that glucose receptor of cAMP-signaling pathway GPCR Gpr1, along with G-protein Gpa2 are essential components of glucose sensing during specific autophagic degradation of peroxisomes (pexophagy).


Fig. 1

Gpr1 and Gpa2 are required for pexophagy in S. cerevisiae. A wild-type (WT; W303-1A), gpa2 (PM731), gpr1 (LK5) and gpr1 gpa2 (LK6) cells were shifted from peroxisome-inducing oleic acid minimal medium to glucose minimal medium without nitrogen source and incubated for 20h. At 0, 10, 15 and 20h of glucose adaptation, protein extracts were prepared and subjected to immunoblot analysis using rabbit antibodies against S. cerevisiae thiolase.


4 Discussion

Yeast cells induce general autophagy during nitrogen and other nutrient deprivations (Klionsky and Ohsumi, 1999; Noda et al., 2002). Special type of autophagy appeared to be the autophagic degradation of peroxisomes or pexophagy (Dunn et al., 2005). In this case, redundant peroxisomes are massively delivered to vacuoles where are degraded by resident proteases. Pexophagy is specific process in terms that inducers of pexophagy are the specific carbon sources, glucose and ethanol (last phenomenon is known only for methanol-induced peroxisomes). Mechanisms of glucose and ethanol sensing in pexophagy process are not known. It was found in the methylotrophic yeast H. polymorpha that glucose sensing in catabolite repression through Gcr1 protein is not involved in pexophagy (Stasyk et al., 2004). Glucose sensing in yeast pexophagy was not studied before the current work. However sensing of this sugar was studied in S. cerevisiae during glucose-specific vacuolar degradation of cytosolic gluconeogenic enzymes fructose-1,6-bisphosphatase and malate dehydrogenase (Hung et al., 2004). It was shown that both Gpr1 and Gpa2 proteins are necessary for degradation of these enzymes. Our data presented in this communication clearly show that both Gpr1 and Gpa2 are indispensable components for glucose-induced autophagic degradation of peroxisomal matrix protein thiolase in S. cerevisiae. So, mechanisms of glucose recognition for autophagic degradation of cytosolic proteins, fructose-1,6-bisphosphatase and malate dehydrogenase (Hung et al., 2004) and peroxisomal protein thiolase (this work) are similar and include G-protein-coupled receptor Gpr1 and interacting with it G-protein Gpa2. Mechanisms of signal transduction from Gpr1 and Gpa2 proteins to autophagic (pexophagic) machinery remain to be elucidated. It is interesting to note that general autophagy (not induced by glucose) in S. cerevisiae is blocked due to constitutive expression of cAMP-signaling pathway (Budovskaya et al., 2004) whereas glucose-specific autophagy is blocked as a result of defect in such a pathway (Hung et al., 2004; this work). It would be interesting to know the effect of constitutive expression of this signaling pathway on glucose-induced autophagy (pexophagy).

Nothing is known on glucose and ethanol sensing participating in pexophagy in methylotrophic yeasts, the most convenient model system for studying pexophagy (Dunn et al., 2005). We plan to isolate mutants defective in orthologs of GPR1 and GPA2 genes in methylotrophic yeasts and to study effect of such mutations on pexophagy of methanol-induced peroxisomes in the medium with glucose. Corresponding work on the model of the methylotrophic yeast Pichia pastoris is in progress.

Acknowledgment

Authors are grateful to Dr. Oleh V. Stasyk (Institute of Cell Biology, NAS of Ukraine) for stimulating discussion.

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Received 23 October 2007; accepted 2 November 2007

doi:10.1016/j.cellbi.2007.11.001


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)