The Ras/cAMP-dependent protein kinase signaling pathway regulates an early step of the autophagy process in Saccharomyces cerevisiae

Abstract

When faced with nutrient deprivation, Saccharomyces cerevisiae cells enter into a nondividing resting state, known as stationary phase. The Ras/PKA (cAMP-dependent protein kinase) signaling pathway plays an important role in regulating the entry into this resting state and the subsequent survival of stationary phase cells. The survival of these resting cells is also dependent upon autophagy, a membrane trafficking pathway that is induced upon nutrient deprivation. Autophagy is responsible for targeting bulk protein and other cytoplasmic constituents to the vacuolar compartment for their ultimate degradation. The data presented here demonstrate that the Ras/PKA signaling pathway inhibits an early step in autophagy because mutants with elevated levels of Ras/PKA activity fail to accumulate transport intermediates normally associated with this process. Quantitative assays indicate that these increased levels of Ras/PKA signaling activity result in an essentially complete block to autophagy. Interestingly, Ras/PKA activity also inhibited a related process, the cytoplasm to vacuole targeting (Cvt) pathway that is responsible for the delivery of a subset of vacuolar proteins in growing cells. These data therefore indicate that the Ras/PKA signaling pathway is not regulating a switch between the autophagy and Cvt modes of transport. Instead, it is more likely that this signaling pathway is controlling an activity that is required during the early stages of both of these membrane trafficking pathways. Finally, the data suggest that at least a portion of the Ras/PKA effects on stationary phase survival are the result of the regulation of autophagy activity by this signaling pathway.

Fig. 1

Elevated levels of Ras/PKA signaling activity inhibited autophagy. A, RAS2^val19 mutants exhibited very low levels of autophagy following nitrogen starvation. The indicated strains were grown to mid-log phase at 30 °C and transferred to the nitrogen starvation medium, SD-N, for 0 or 15 h. Autophagy levels were measured with the alkaline phosphatase-based assay as described under “Experimental Procedures.” The levels of autophagy induction are indicated by the relative increase in alkaline phosphatase activity that is induced by the nitrogen starvation. The strains used were wild type (TN125), atg1Δ (YYK126), atg13Δ (YYK130), and RAS2^val19 (PHY3513). All strains except PHY3513 carried the control vector, pRS413. B, increased levels of PKA activity inhibited autophagy activity. Alkaline phosphatase assays were performed as described above in A. The strains used were wild-type (TN125 with the control vector, pRS426), HC-TPK1 (wild-type carrying the high-copy number TPK1 plasmid, pPHY2056) and atg1Δ (YYK126 with pRS426). C, elevated levels of Ras/PKA signaling activity inhibited the rapamycin-mediated induction of autophagy. The indicated strains were grown to mid-log phase in YM-glucose medium and then treated with 0.2 μg/ml rapamycin for 0, 1, or 3 h. Autophagy levels were then assessed with the alkaline phosphatase-based assay system. The strains analyzed were those used in A.

Fig. 2

Inactivation of the Ras/PKA signaling pathway was sufficient to induce an autophagy response. A, expression of the dominant negative RAS2^ala22 allele resulted in a growth arrest after transfer to the galactose medium. Wild-type cells (TN125) carrying either a control vector, pRS416 (circles), or the GAL1-RAS2^ala22 plasmid, pPHY2128 (triangles), were grown to mid-log phase in YM-glucose medium and transferred to a YM medium that contained 5% galactose and 2% raffinose. The culture density was assessed by spectrophoto-metric measurement of the OD₆₀₀ at the indicated times after the transfer to galactose-containing growth medium. B, inactivation of the Ras/PKA pathway resulted in an induction of autophagy activity. Autophagy levels were assessed with the alkaline phosphatase-based assay as described under “Experimental Procedures.”

Fig. 3

Elevated levels of Ras/PKA signaling activity blocked the formation of autophagy pathway intermediates. The pep4Δ (TVY1) and vam3^ts (TDY2) strains carrying either the MET3-RAS2^val19plasmid, pPHY446, or a control vector, pRS413, were grown to mid-log phase and transferred to nitrogen starvation medium for 3 h. All incubations with the pep4Δ strains were performed at 30 °C. The vam3^ts strain was grown initially at 26 °C but the incubation in starvation medium was performed at 37 °C so as to inactivate the thermolabile Vam3p present. The cells were then collected and processed for electron microscopy as described under “Experimental Procedures.” The arrows in the figure indicate the autophagic bodies accumulating in the vacuoles of the pep4Δ strain, and the arrowheads indicate the autophagosomes present in the cytoplasm of the vam3^ts strain. Note that both of these structures are absent in cells expressing the RAS2^val19 allele. The bar indicates 1 μm.

Fig. 4

Elevated levels of Ras/PKA signaling activity inhibited the expression and processing of the Cvt cargo protein, Ape1p. A, levels of Ape1p were greatly diminished in RAS2^val19 mutants. Protein extracts were prepared from mid-log phase cultures of the indicated strains. The levels of Ape1p present in these extracts were assessed by a Western immunoblot with a polyclonal antiserum specific for this protein. The positions of the mature (Ape1p) and precursor (prApe1p) forms of Ape1p are shown. The relative levels of a cytoplasmic protein, Pgk1p, were assessed to ensure that similar amounts of protein were present in each sample. The strains analyzed were wild-type (TN125), RAS2^val19 (PHY3513), atg1Δ (YYK126), and atg13Δ (YYK130). All strains except PHY3513 contained the control vector, pRS413. The RAS2^val19 strain was grown in medium that either lacked (RAS2^val19 (on)) or contained 500 μm methionine (RAS2^val19 (off)); the other strains were grown in the medium lacking methionine. B, elevated levels of Ras/PKA signaling activity inhibited the processing of Ape1p. Strains with the indicated genotypes were transformed with a plasmid containing the CUP1-APE1 construct. These strains were grown to mid-log phase in YM-glucose medium and the relative levels of the two forms of Ape1p were assessed by Western immunoblotting. The strains analyzed were as in A. C, elevated levels of Ras/PKA signaling activity did not affect the processing of the vacuolar hydrolase, CPY. The processing of CPY was assessed as described under “Experimental Procedures.” Briefly, yeast strains were grown to mid-log phase in YM-glucose medium and labeled with a [³⁵S]methionine/cysteine mixture for 20 min at 30 °C. An excess of unlabeled amino acids was added, and this chase incubation was continued at 30 °C for an additional 30 min. Protein extracts were prepared and incubated with an antibody specific for CPY. The immunoprecipitated proteins were separated on a 7.5% SDS-PAGE gel that was subsequently subjected to autoradiography. The positions of the p2 precursor (p2 CPY) and mature (CPY) forms of CPY are indicated. The strains analyzed were as in A.

Fig. 5

The starvation-induced processing of Ape1p was inhibited by Ras/PKA signaling activity. The CUP1-APE1 construct was introduced into strains with the indicated genotypes. The strains were grown to mid-log phase at 30 °C in a YM-glucose medium containing 100 μm CuSO₄ to induce high level expression from the CUP1 promoter. The cultures were then transferred to the nitrogen starvation medium, SD-N, and incubated at 30°C for 3, 7, or 15 h. Protein extracts were prepared from these cultures, and the relative levels of Ape1p processing were assessed with a Western immunoblot using a polyclonal antiserum specific for Ape1p. The relative processing at each starvation point (S) was compared with that observed in the mid-log phase, or nonstarved, control culture (N). The positions of the mature and precursor forms of Ape1p are shown. The strains analyzed were wild-type (TN125), RAS2^val19 (PHY3513), atg1Δ (YYK126), and atg13Δ (YYK130). All strains except PHY3513 also carried the control vector, pRS413.

Fig. 6

The rapamycin-induced dephosphorylation of Atg13p was not inhibited by the presence of the RAS2^val19 allele. Wild-type and RAS2^val19 cells carrying a high-copy ATG13 plasmid were grown to mid-log phase and then treated with 0.2 μg/ml rapamycin for 0 or 60 min. Protein extracts were prepared and the relative mobility of Atg13p in an SDS-polyacrylamide gel was assessed by Western immunoblotting with a polyclonal antiserum specific for Atg13p. The positions of the hyperphosphorylated forms of Atg13p found in log phase cells are indicated (pp-Atg13p). The strains analyzed were wild-type (TN125 with the control vector, pRS413) and RAS2^val19 (PHY3513). The strains were grown in either medium lacking methionine (wild-type and RAS2^val19 (on)) or containing 500 μm methionine (RAS2^val19 (off)).

Fig. 7

Potential models to describe how Ras/PKA signaling activity influences autophagy and how this control might be coordinated with that of the Tor signaling pathway. A, Ras/PKA signaling pathway regulates an early event in autophagy, an event that precedes the formation of the autophagosome. The schematic shows a number of the steps involved in the autophagy process and the relative position of the event most likely to be controlled by Ras/PKA signaling activity. B, schematic depicting the relative order of action of the Ras/PKA and Tor signaling pathways on the autophagy process. The data presented here, and in other recent studies, are consistent with the Ras/PKA pathway acting either (i) independently or (ii) downstream of the Tor signaling pathway. See the text for further details.