C. Randell Brown

Cyclophilin A Mediates Vid22p Function in the Import of Fructose-1,6-bisphosphatase into Vid Vesicles

Fructose-1,6-bisphosphatase (FBPase) is synthesized in yeast during glucose starvation but is rapidly degraded in the vacuole following the addition of glucose. FBPase trafficking to the vacuole involves two distinct steps, import into intermediate transport vesicles (Vid vesicles) and Vid vesicle trafficking to the vacuole. FBPase import into Vid vesicles requires the VID22gene. However, VID22 affects FBPase import indirectly through a cytosolic factor. To identify the required cytosolic component, wild type cytosol was fractionated and screened for proteins that complement Δvid22 mutant cytosol using an in vitro assay that reproduces FBPase import into Vid vesicles. Cyclophilin A (Cpr1p) was identified as a cytosolic protein that mediates Vid22p function in FBPase import. Mutants lacking Cpr1p were defective in FBPase import. Furthermore, the addition of purified Cpr1p restored FBPase import in both the Δcpr1 and the Δvid22 mutants. The cyclosporin A binding pocket is important for Cpr1p function, since cyclosporin A binding-deficient mutants failed to complement FBPase import in Δcpr1 and Δvid22 mutants. The levels of Cpr1p were reduced in the Δvid22 mutants, implying that the expression of Cpr1p is regulated by Vid22p. Our results suggest that Cpr1p mediates Vid22p function and is directly involved in the import of FBPase into Vid vesicles.

Alcohol regulates eukaryotic elongation factor 2 phosphorylation via an AMP-activated protein kinase-dependent mechanism in C2C12 skeletal myocytes.

Ethanol decreases protein synthesis in cells, although the underlying regulatory mechanisms of this process are not fully established. In the present study incubation of C2C12 myocytes with 100 mm EtOH decreased protein synthesis while markedly increasing the phosphorylation of eukaryotic elongation factor 2 (eEF2), a key component of the translation machinery. Both mTOR and MEK pathways were found to play a role in regulating the effect of EtOH on eEF2 phosphorylation. Rapamycin, an inhibitor of mammalian target of rapamycin, and the MEK inhibitor PD98059 blocked the EtOH-induced phosphorylation of eEF2, whereas the p38 MAPK inhibitor SB202190 had no effect. Unexpectedly, EtOH decreased the phosphorylation and activity of the eEF2 upstream regulator eEF2 kinase. Likewise, treatment of cells with the inhibitor rottlerin did not block the stimulatory effect of EtOH on eEF2, suggesting that eEF2 kinase (eEF2K) does not play a role in regulating eEF2. In contrast, increased eEF2 phosphorylation was correlated with an increase in AMP-activated protein kinase (AMPK) phosphorylation and activity. Compound C, an inhibitor of AMPK, suppressed the effects of EtOH on eEF2 phosphorylation but had no effect on eEF2K, indicating that AMPK regulates eEF2 independent of eEF2K. Finally, EtOH decreased protein phosphatase 2A activity when either eEF2 or AMPK was used as the substrate. Thus, this later action may partially account for the increased phosphorylation of eEF2 in response to EtOH and the observed sensitivity of AMPK to rapamycin and PD98059 treatments. Collectively, the induction of eEF2 phosphorylation by EtOH is controlled by an increase in AMPK and a decrease in protein phosphatase 2A activity.

Alcohol and PRAS40 Knockdown Decrease mTOR Activity and Protein Synthesis via AMPK Signaling and Changes in mTORC1 Interaction

The mTORC1 protein kinase complex consists of mTOR, raptor, mLST8/GβL and PRAS40. Previously, we reported that mTOR plays an important role in regulating protein synthesis in response to alcohol (EtOH). However, the mechanisms by which EtOH regulates mTORC1 activity have not been established. Here, we investigated the effect of EtOH on the phosphorylation and interaction of components of mTORC1 in C2C12 myocytes. We also examined the specific role that PRAS40 plays in this process. Incubation of myocytes with EtOH (100 mM, 24 h) increased raptor and PRAS40 phosphorylation. Likewise, there were increased levels of the PRAS40 upstream regulators Akt and IRS‐1. EtOH also caused changes in mTORC1 protein–protein interactions. EtOH enhanced the binding of raptor and PRAS40 with mTOR. These alterations occurred in concert with increased binding of 14‐3‐3 to raptor, while the PRAS40 and 14‐3‐3 interaction was not affected. The shRNA knockdown (KD) of PRAS40 decreased protein synthesis similarly to EtOH. PRAS40 KD increased raptor phosphorylation and its association with 14‐3‐3, whereas decreased GβL–mTOR binding. The effects of EtOH and PRAS40 KD were mediated by AMPK. Both factors increased in vitro AMPK activity towards the substrate raptor. In addition, KD enhanced the activity of AMPK towards TSC2. Collectively, our results indicate that EtOH stabilizes the association of raptor, PRAS40, and GβL with mTOR, while likewise increasing the interaction of raptor with 14‐3‐3. These data suggest a possible mechanism for the inhibitory effects of EtOH on mTOR kinase activity and protein synthesis in myocytes. J. Cell. Biochem. 109: 1172–1184, 2010.

The Vacuolar Import and Degradation Pathway Merges with the Endocytic Pathway to Deliver Fructose-1,6-bisphosphatase to the Vacuole for Degradation

The gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is degraded in the vacuole when glucose is added to glucose-starved cells. Before it is delivered to the vacuole, however, FBPase is imported into intermediate carriers called Vid (vacuole import and degradation) vesicles. Here, using biochemical and genetic approaches, we identified a requirement for SEC28 in FBPase degradation. SEC28 encodes the ϵ-COP subunit of COPI (coat protein complex I) coatomer proteins. When SEC28 and other coatomer genes were mutated, FBPase degradation was defective and FBPase association with Vid vesicles was impaired. Coatomer proteins were identified as components of Vid vesicles, and they formed a protein complex with a Vid vesicle-specific protein, Vid24p. Furthermore, Vid24p association with Vid vesicles was impaired when coatomer genes were mutated. Kinetic studies indicated that Sec28p traffics to multiple locations. Sec28p was in Vid vesicles, endocytic compartments, and the vacuolar membrane in various mutants that block the FBPase degradation pathway. Sec28p was also found in vesicles adjacent to the vacuolar membrane in the ret2-1 coatomer mutant. We propose that Sec28p resides in Vid vesicles, and these vesicles converge with the endocytic pathway. After fusion, Sec28p is distributed on the vacuolar membrane, where it concentrates on vesicles that pinch off from this organelle. FBPase also utilizes the endocytic pathway for transport to the vacuole, as demonstrated by its presence in endocytic compartments in the Δvph1 mutant. Taken together, our results indicate a strong connection between the Vid trafficking pathway and the endocytic pathway.

The TOR complex 1 is distributed in endosomes and in retrograde vesicles that form from the vacuole membrane and plays an important role in the vacuole import and degradation pathway

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to cells that have been starved for 3 days, FBPase is degraded in the vacuole. FBPase is first imported to Vid (vacuole import and degradation) vesicles, and these vesicles then merge with the endocytic pathway. In this report we show that two additional gluconeogenic enzymes, isocitrate lyase and phosphoenolpyruvate carboxykinase, were also degraded in the vacuole via the Vid pathway. These new cargo proteins and FBPase interacted with the TORC1 complex during glucose starvation. However, Tor1p was dissociated from FBPase after the addition of glucose. FBPase degradation was inhibited in cells overexpressing TOR1, suggesting that excessive Tor1p is inhibitory. Both Tco89p and Tor1p were found in endosomes coming from the plasma membrane as well as in retrograde vesicles forming from the vacuole membrane. When TORC1 was inactivated by rapamycin, FBPase degradation was inhibited. We suggest that TORC1 interacts with multiple cargo proteins destined for the Vid pathway and plays an important role in the degradation of FBPase in the vacuole.

Rag GTPases and AMPK/TSC2/Rheb mediate the differential regulation of mTORC1 signaling in response to alcohol and leucine

Leucine (Leu) and insulin both stimulate muscle protein synthesis, albeit at least in part via separate signaling pathways. While alcohol (EtOH) suppresses insulin-stimulated protein synthesis in cultured myocytes, its ability to disrupt Leu signaling and Rag GTPase activity has not been determined. Likewise, little is known regarding the interaction of EtOH and Leu on the AMPK/TSC2/Rheb pathway. Treatment of myocytes with EtOH (100 mM) decreased protein synthesis, whereas Leu (2 mM) increased synthesis. In combination, EtOH suppressed the anabolic effect of Leu. The effects of EtOH and Leu were associated with coordinate changes in the phosphorylation state of mTOR, raptor, and their downstream targets 4EBP1 and S6K1. As such, EtOH suppressed the ability of Leu to activate these signaling components. The Rag signaling pathway was activated by Leu but suppressed by EtOH, as evidenced by changes in the interaction of Rag proteins with mTOR and raptor. Overexpression of constitutively active (ca)RagA and caRagC increased mTORC1 activity, as determined by increased S6K1 phosphorylation. Furthermore, the caRagA-caRagC heterodimer blocked the inhibitory effect of EtOH. EtOH and Leu produced differential effects on AMPK signaling. EtOH enhanced AMPK activity, resulting in increased TSC2 (S1387) and eEF2 phosphorylation, whereas Leu had the opposite effect. EtOH also decreased the interaction of Rheb with mTOR, and this was prevented by Leu. Collectively, our results indicate that EtOH inhibits the anabolic effects that Leu has on protein synthesis and mTORC1 activity by modulating both Rag GTPase function and AMPK/TSC2/Rheb signaling.

The vacuole import and degradation pathway utilizes early steps of endocytosis and actin polymerization to deliver cargo proteins to the vacuole for degradation

When glucose is added to yeast cells that are starved for 3 days, fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase 2 are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. In this study, we examined the distribution of FBPase at the ultrastructural level. FBPase was observed in areas close to the plasma membrane and in cytoplasmic structures that are heterogeneous in size and density. We have isolated these intracellular structures that contain FBPase, the Vid vesicle marker Vid24p, and the endosomal marker Pep12p. They appeared irregular in size and shape. In yeast, actin polymerization plays an important role in early steps of endocytosis. Mutants that affect actin polymerization inhibited FBPase degradation, suggesting that actin polymerization is important for FBPase degradation. Both FBPase and malate dehydrogenase 2 were associated with actin patches. Vid vesicle proteins such as Vid24p or Sec28p were also at actin patches, although they dissociated from these structures at later time points. We propose that Vid24p and Sec28p are present at actin patches during glucose starvation. Cargo proteins arrive at these sites following the addition of glucose, and the endocytic vesicles then pinch off from the plasma membrane. Following the fusion of endosomes with the vacuole, cargo proteins are then degraded in the vacuole.

Alcohol-Induced Modulation of Rictor and mTORC2 Activity in C2C12 Myoblasts

BACKGROUND The mammalian target of rapamycin (mTOR) kinase controls cell growth, proliferation, and metabolism through 2 distinct multiprotein complexes, mTORC1 and mTORC2. We reported that alcohol (EtOH) inhibits mTORC1 activity and protein synthesis in C2C12 myoblasts. However, the role that mTORC2 plays in this process has not been elucidated. In this study, we investigated whether mTORC2 functions as part of a feedback regulator in response to EtOH, acting to maintain the balance between the functions of Akt, mTORC2, and mTORC1. METHODS C2C12 myoblasts were incubated with EtOH for 18 to 24 hours. Levels of various mTORC2 proteins and mRNA were assessed by immunoblotting and real-time PCR, respectively, while protein-protein interactions were determined by immunoprecipitation and immunoblotting. An in vitro mTORC2 kinase activity assay was performed using Akt as a substrate. The rate of protein synthesis was determined by (35) S-methionine/cysteine incorporation into cellular protein. RESULTS EtOH (100 mM) increased the protein and mRNA levels of the mTORC2 components rictor, mSin1, proline-rich repeat protein 5, and Deptor. There was also an increased association of these proteins with mTOR. EtOH increased the in vitro kinase activity of mTORC2, and this was correlated with decreased binding of rictor with 14-3-3 and Deptor. Reduced rictor phosphorylation at T1135 by EtOH was most likely due to decreased S6K1 activity. Knockdown of rictor elevated mTORC1 activity, as indicated by increased S6K1 phosphorylation and protein synthesis. Likewise, there were decreased amounts and/or phosphorylation levels of various mTORC1 and mTORC2 components including raptor, proline-rich Akt substrate 40 kDa, mSin1, Deptor, and GβL. Activated PP2A was associated with decreased Akt and eukaryotic elongation factor 2 phosphorylation. Collectively, our results provide evidence of a homeostatic balance between the 2 mTOR complexes following EtOH treatments in myoblasts. CONCLUSIONS   EtOH increased the activity of mTORC2 by elevating levels of various components and their interaction with mTOR. Decreased rictor phosphorylation at T1135 acts as mTORC1-dependent feedback mechanisms, functioning in addition to the insulin receptor substrate-I/PI3K signaling pathway to regulate protein synthesis.

Lopinavir Impairs Protein Synthesis and Induces eEF2 Phosphorylation via the Activation of AMP-Activated Protein Kinase

HIV anti‐retroviral drugs decrease protein synthesis, although the underlying regulatory mechanisms of this process are not fully established. Therefore, we investigated the effects of the HIV protease inhibitor lopinavir (LPV) on protein metabolism. We also characterized the mechanisms that mediate the effects of this drug on elongation factor‐2 (eEF2), a key component of the translational machinery. Treatment of C2C12 myocytes with LPV produced a dose‐dependent inhibitory effect on protein synthesis. This effect was observed at 15 min and was maintained for at least 4 h. Mechanistically, LPV increased the phosphorylation of eEF2 and thereby decreased the activity of this protein. Increased phosphorylation of eEF2 was associated with increased activity of its upstream regulators AMP‐activated protein kinase (AMPK) and eEF2 kinase (eEF2K). Both AMPK and eEF2K directly phosphorylated eEF2 in an in vitro kinase assay suggesting two distinct paths lead to eEF2 phosphorylation. To verify this connection, myocytes were treated with the AMPK inhibitor compound C. Compound C blocked eEF2K and eEF2 phosphorylation, demonstrating that LPV affects eEF2 activity via an AMPK‐eEF2K dependent pathway. In contrast, incubation of myocytes with rottlerin suppressed eEF2K, but not eEF2 phosphorylation, suggesting that eEF2 can be regulated independent of eEF2K. Finally, LPV did not affect PP2A activity when either eEF2 or peptide was used as the substrate. Collectively, these results indicate that LPV decreases protein synthesis, at least in part, via inhibition of eEF2. This appears regulated by AMPK which can act directly on eEF2 or indirectly via the action of eEF2K. J. Cell. Biochem. 105: 814–823, 2008.

Degradation of the gluconeogenic enzyme fructose-1, 6-bisphosphatase is dependent on the vacuolar ATPase.

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced during glucose starvation. After the addition of glucose, inactivated FBPase is selectively targeted to a novel type of Vid (vacuolar import and degradation) vesicle and then to the vacuole for degradation. To identify proteins involved in this pathway, we screened various libraries for mutants that failed to degrade FBPase. Via these approaches, subunits of the vacuolar H+ ATPase (V-ATPase) have been identified repeatedly. The VATPase has established roles in endocytosis, sorting of carboxypeptidase Y and homotypic vacuole fusion. Here, we show that Stv1p, Vph1p, and other subunits of the VATPase are required for FBPase degradation. VPH1 and V0 domain subunits such as Vma3p were required for both Vid vesicle and vacuole function, as determined by an in vitro fusion assay. However, STV1 was only required for the proper function of the Vid vesicles. We also show that the V1 domain participates in the Vid vesicle to vacuoletrafficking step, since most of the V1 subunits are necessary for Vid vesicle-vacuole fusionto occur. The V0 and V1 domains are assembled following a glucose shift and theassembly is independent of protein kinase A and RAV genes. Assembly of the V0 complexis necessary for FBPase trafficking, since mutants that block the assembly and transport ofV0 out of the ER were defective in FBPase degradation.