FKBP12-Rapamycin-associated Protein or Mammalian Target of Rapamycin (FRAP/mTOR) Localization in the Endoplasmic Reticulum and the Golgi Apparatus

Abstract

FKBP12-rapamycin-associated protein (FRAP) or mammalian target of rapamycin (mTOR) and its effector proteins form a critical signaling pathway that regulates eukaryotic cell growth and proliferation. Although the protein components in this pathway have begun to be identified, little is known about their subcellular localization or the physiological significance of their localization. By immunofluorescence, we find that both endogenous and recombinant FRAP/mTOR proteins show localization predominantly in the endoplasmic reticulum (ER) and the Golgi apparatus. Consistent with this finding, FRAP/mTOR is cofractionated with calnexin, an ER marker protein. Biochemical characterization suggests that FRAP/mTOR is a peripheral ER/Golgi protein with tight membrane association. Finally, we have identified domains of FRAP/mTOR which may mediate its association with the ER and the Golgi apparatus. FKBP12-rapamycin-associated protein (FRAP) or mammalian target of rapamycin (mTOR) and its effector proteins form a critical signaling pathway that regulates eukaryotic cell growth and proliferation. Although the protein components in this pathway have begun to be identified, little is known about their subcellular localization or the physiological significance of their localization. By immunofluorescence, we find that both endogenous and recombinant FRAP/mTOR proteins show localization predominantly in the endoplasmic reticulum (ER) and the Golgi apparatus. Consistent with this finding, FRAP/mTOR is cofractionated with calnexin, an ER marker protein. Biochemical characterization suggests that FRAP/mTOR is a peripheral ER/Golgi protein with tight membrane association. Finally, we have identified domains of FRAP/mTOR which may mediate its association with the ER and the Golgi apparatus. Rapamycin is a macrolide isolated from the Easter Island soil bacterium Streptomyces hygroscopicus and is currently used as an immunosuppressant in kidney transplantation (1.Sehgal S.N. Ther. Drug Monit. 1995; 17: 660-665Crossref PubMed Scopus (259) Google Scholar) and in drug-eluting stents to prevent restenosis after angioplasty (2.Marx S.O. Marks A.R. Circulation. 2001; 104: 852-855Crossref PubMed Scopus (358) Google Scholar). In addition, rapamycin ester derivatives are in clinical trials for cancer treatment (3.Huang S. Houghton P. Curr. Opin. Investig. Drugs. 2002; 3: 295-304PubMed Google Scholar). The FKBP12-rapamycin-associated protein (FRAP), 1The abbreviations used are: FRAP, FKBP12-rapamycin-associated protein; DAPI, 4,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; GST, glutathione S-transferase; IF, immunofluorescence; MOPS, 4-morpholinepropanesulfonic acid; mTOR, mammalian target of rapamycin; S6K(s), S6 kinase(s); SREBP, sterol response element-binding protein. also known as mammalian target of rapamycin (mTOR), is a 289-kDa phosphatidylinositol 3-kinase-related kinase (4.Hunter T. Cell. 1995; 83: 1-4Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 5.Keith C.T. Schreiber S.L. Science. 1995; 270: 50-51Crossref PubMed Scopus (448) Google Scholar). The phosphatidylinositol 3-kinase-related kinase family also includes ATM, ATR, DNA-PK, and SMG-1. FKBP12-rapamycin binds to and inhibits FRAP in mammalian cells and its homolog Tor1/2 in yeast via the conserved FKBP12-rapamycin binding domain (6.Chen J. Zheng X.F. Brown E.J. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4947-4951Crossref PubMed Scopus (448) Google Scholar, 7.Choi J. Chen J. Schreiber S.L. Clardy J. Science. 1996; 273: 239-242Crossref PubMed Scopus (723) Google Scholar, 8.Stan R. McLaughlin M.M. Cafferkey R. Johnson R.K. Rosenberg M. Livi G.P. J. Biol. Chem. 1994; 269: 32027-32030Abstract Full Text PDF PubMed Google Scholar, 9.Zheng X.F. Florentino D. Chen J. Crabtree G.R. Schreiber S.L. Cell. 1995; 82: 121-130Abstract Full Text PDF PubMed Scopus (246) Google Scholar). The C-terminal kinase domain is homologous to phosphatidylinositol 3-kinase catalytic domains, although it only appears to have protein kinase activity (4.Hunter T. Cell. 1995; 83: 1-4Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 5.Keith C.T. Schreiber S.L. Science. 1995; 270: 50-51Crossref PubMed Scopus (448) Google Scholar). At the N terminus, there is an extensive array of tandem HEAT repeats (10.Perry J. Kleckner N. Cell. 2003; 112: 151-155Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 11.Andrade M.A. Bork P. Nat. Genet. 1995; 11: 115-116Crossref PubMed Scopus (466) Google Scholar) that mediate protein-protein interactions with CLIP-170, Gephyrin and Raptor (12.Choi J.H. Bertram P.G. Drenan R. Carvalho J. Zhou H.H. Zheng X.F.S. EMBO Rep. 2002; 3: 988-994Crossref PubMed Scopus (88) Google Scholar, 13.Hara K. Maruki Y. Long X. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1448) Google Scholar, 14.Kim D. Sarbassov d.D. Ali S. King J. Latek R. Erdjument-Bromage H. Tempst P. Sabatini D. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2371) Google Scholar, 15.Sabatini D.M. Barrow R.K. Blackshaw S. Burnett P.E. Lai M.M. Field M.E. Bahr B.A. Kirsch J. Betz H. Snyder S.H. Science. 1999; 284: 1161-1164Crossref PubMed Scopus (160) Google Scholar). Two other domains conserved in the phosphatidylinositol 3-kinase-related kinase family, FAT and FATC, flank the kinase domain to the N and C terminus, respectively (16.Bossini R. Isacchi A. Sonnhammer E.L. Trends Biochem. Sci. 2000; 25: 225-227Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). FRAP is linked to translational control through its two main effector proteins, the ribosomal S6 kinases (S6Ks) and the translation regulator eIF4E-binding protein 1 (17.Dennis P.B. Fumagalli S. Thomas G. Curr. Opin. Genet. Dev. 1999; 9: 49-54Crossref PubMed Scopus (249) Google Scholar, 18.Kuruvilla F. Schreiber S.L. Chem. Biol. 1999; 6: R129-R136Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 19.Rohde J. Heitman J. Cardenas M. J. Biol. Chem. 2001; 276: 7027-7036Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 20.Raught B. Gingras A.-C. Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7037-7044Crossref PubMed Scopus (507) Google Scholar, 21.Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1729) Google Scholar). S6Ks promote protein synthesis by phosphorylation of ribosomal protein S6, allowing the translation of 5′-oligopyrimidine tract-containing mRNAs that encode for ribosomal proteins and translational regulators (17.Dennis P.B. Fumagalli S. Thomas G. Curr. Opin. Genet. Dev. 1999; 9: 49-54Crossref PubMed Scopus (249) Google Scholar, 18.Kuruvilla F. Schreiber S.L. Chem. Biol. 1999; 6: R129-R136Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 19.Rohde J. Heitman J. Cardenas M. J. Biol. Chem. 2001; 276: 7027-7036Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 20.Raught B. Gingras A.-C. Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7037-7044Crossref PubMed Scopus (507) Google Scholar, 21.Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1729) Google Scholar). eIF4E-binding protein 1 binds to eIF4E and inhibits CAP-dependent translation under unfavorable growth conditions. eIF4E-binding protein 1 becomes phosphorylated by FRAP and is released from eIF4E when growth conditions become favorable (17.Dennis P.B. Fumagalli S. Thomas G. Curr. Opin. Genet. Dev. 1999; 9: 49-54Crossref PubMed Scopus (249) Google Scholar, 18.Kuruvilla F. Schreiber S.L. Chem. Biol. 1999; 6: R129-R136Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 19.Rohde J. Heitman J. Cardenas M. J. Biol. Chem. 2001; 276: 7027-7036Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 20.Raught B. Gingras A.-C. Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7037-7044Crossref PubMed Scopus (507) Google Scholar, 21.Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1729) Google Scholar). FRAP also controls protein synthesis by regulating ribosome biogenesis (22.Power T. Walter P. Mol. Biol. Cell. 1999; 10: 987-1000Crossref PubMed Scopus (325) Google Scholar, 23.Tsang C.K. Bertram P.G. Ai W. Drenan R. Zheng X.F. EMBO J. 2003; 22: 6045-6056Crossref PubMed Scopus (137) Google Scholar, 24.Zaragoza D. Ghavidel A. Heitman J. Schultz M.C. Mol. Cell. Biol. 1998; 18: 4463-4470Crossref PubMed Scopus (184) Google Scholar). FRAP signaling is regulated both by nutrients, including amino acids and glucose, and by mitogens (17.Dennis P.B. Fumagalli S. Thomas G. Curr. Opin. Genet. Dev. 1999; 9: 49-54Crossref PubMed Scopus (249) Google Scholar, 18.Kuruvilla F. Schreiber S.L. Chem. Biol. 1999; 6: R129-R136Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 19.Rohde J. Heitman J. Cardenas M. J. Biol. Chem. 2001; 276: 7027-7036Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 20.Raught B. Gingras A.-C. Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7037-7044Crossref PubMed Scopus (507) Google Scholar, 21.Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1729) Google Scholar). Both the endoplasmic reticulum (ER) and the Golgi apparatus are internal membrane structures constituting more than one-half of the total membranes in a cell. The ER is known to have two essential functions. First, proteins destined for transport to other organelles and the cell surface or for secretion are synthesized in the ER. During translation, they are translocated into the ER lumen through a pore in the ER membrane and are folded and become glycosylated inside the ER lumen. Secondly, synthesis of lipids and cholesterol takes place on the cytoplasmic side of the ER membrane. The ER is also an important participant in cell signaling, including the unfolded protein response, the ER overload response, and the ER-to-nuclear sterol signaling by SREBP (25.Pahl H.L. Physiol. Rev. 1999; 79: 683-701Crossref PubMed Scopus (307) Google Scholar). Several recent studies suggest that the ER is also involved in signal transduction pathways traditionally thought to occur solely through the plasma membrane. For example, the small GTPase Ras that is restricted to the ER and the Golgi apparatus can actively engage in signal transduction to mitogen-activated protein kinases (26.Chiu V. Bivona T. Hach A. Sajous J. Silletti J. Wiener H. Johnson R. Cox A. Philips M. Nat. Cell Biol. 2002; 4: 343-350Crossref PubMed Scopus (517) Google Scholar). These studies indicate that the ER and the Golgi apparatus can serve as anchors for diverse cell signaling events. A large body of evidence shows that proper localization plays a crucial role in the specificity and/or regulation of cell signaling events. Despite the importance of FRAP in cell growth and therapeutic intervention, little is known about its precise subcellular localization. We have examined common mammalian cell lines for FRAP localization. We find that FRAP is localized predominantly in the ER and the Golgi apparatus. Biochemical analyses suggest that FRAP is a peripheral ER membrane protein but tightly anchored to the ER/Golgi membranes. Finally, we found two discrete domains in the N terminus which are crucial for targeting to the ER and the Golgi apparatus, respectively. Like the sterol-SREBP pathway, FRAP may use the ER/Golgi as anchors for nutrient sensing and signaling. Plasmids, Cell Lines, Antibodies, and Chemicals—FLAG-FRAP plasmids have been described previously (12.Choi J.H. Bertram P.G. Drenan R. Carvalho J. Zhou H.H. Zheng X.F.S. EMBO Rep. 2002; 3: 988-994Crossref PubMed Scopus (88) Google Scholar). Mammalian cell lines were obtained from American Type Culture Center (ATCC) and were maintained as recommended by ATCC. FRAP antibodies were produced (Harlan) by immunizing rabbits with polypeptides corresponding to the amino acids 1-120 of human FRAP. The rabbit FLAG polyclonal antibody was purchased from Sigma, calnexin antibodies were purchased from BD Transduction Laboratories, Golgin-97 antibodies and Mitotracker were purchased from Molecular Probes, phospho-Thr389 antibody was purchased from Cell Signaling, FRAP H-266 FKBP12-rapamycin binding-specific antibody was purchased from Santa Cruz Biotechnology, and horseradish peroxidase-conjugated secondary antibodies were purchased from Pierce. Leptomycin B, sodium azide, and trypsin and chymotrypsin were purchased from Sigma. Affinity Purification of FRAP Antibodies—The N-terminal FRAP antigenic polypeptide was conjugated to CNBr-activated Sepharose beads (Amersham Biosciences) according to manufacturer's instruction. The N-terminal FRAP antiserum (150 mg of IgG) was equilibrated in binding buffer (Bio-Rad) and was loaded twice onto the N-terminal FRAP-Sepharose affinity column. After washing with binding buffer, FRAP-specific IgG was eluted with elution buffer (Bio-Rad). Fractions were collected, analyzed, and subsequently dialyzed against 10 mm HEPES, pH 7.5, 150 mm NaCl, 50% glycerol until further use. Purification and dialysis were performed at 4 °C. Immunoprecipitation and Western Blot—Cell lysates for immunoprecipitation and Western blot were prepared using ice-cold lysis buffer containing 50 mm HEPES-KOH, pH 7.4, 40 mm NaCl, 1 mm EDTA, 0.1% Triton X-100, 5% glycerol, 10 mm sodium pyrophosphate, 10 mm β-glycerophosphate, 1.5 mm Na3VO4, 50 mm NaF, and 1× protease inhibitor mixture (Roche Diagnostics, GmbH). Immunoprecipitation was performed by incubating the cell lysates with 10 μl of FRAP antibody/mg of protein at 4 for The were to protein beads (Amersham Biosciences) for 1 at 4 which were with buffer containing 50 mm HEPES-KOH, pH 7.4, 1 0.1% Triton X-100, 5% glycerol, 10 mm sodium pyrophosphate, 10 mm β-glycerophosphate, 1.5 mm Na3VO4, 50 mm NaF, and 1× protease inhibitor were eluted by in protein For Western protein were on and onto an membrane. After with 5% in the membrane was with antibodies for 1 to by with horseradish peroxidase-conjugated secondary antibodies for and with (Amersham FRAP FRAP or control were at with the FRAP kinase mm HEPES-KOH, pH 7.4, 10 mm 1 mm mm of μl of kinase of were at and the kinase was by in of at was by Western blot with a phospho-Thr389 and cells were with plasmids using the For IF, cells were in in for 10 at with ice-cold Triton buffer Triton in mm HEPES, pH 7.4, 50 mm NaCl, mm mm for on with 0.1% in for 10 on and with antibodies for N-terminal FRAP antibody at rabbit C-terminal FRAP antibody at FRAP H-266 FKBP12-rapamycin binding-specific antibody at at at rabbit at at in a antibodies were by washing 10 with antibodies with or were for at and as with the cells were with onto and using a and or an with a in was with the N-terminal FRAP antiserum or N-terminal FRAP Mitotracker was used to cells according to manufacturer's and cells were with ice-cold buffer mm HEPES, mm mm EDTA, pH in of ice-cold buffer containing mm and with 10 and cells were at by a at 4 The were in The an was on a and further at 4 The were in The was also for Western blot the of FRAP membrane the were in 4 in 10 mm MOPS, or sodium pH and for 40 at 4 to Two were performed for For protease the were in 10 mm with or Triton X-100, and with of for 40 at 4 °C. were by the of and in protein FRAP subcellular we have a polyclonal antibody against the N-terminal amino acids of FRAP. antibody a protein of in cell including and human A of was which appears to be a FRAP cell Western blot signal was by the FRAP antigenic polypeptide and this Western blot signal was when a was used In addition, the polypeptide was by the FRAP but a further the specificity of the FRAP we the of the to using a by Thomas and P.B. A. M. B. Thomas G. Science. 2001; PubMed Scopus Google Scholar). In this the was with recombinant acids and a of at a phosphorylation was by Western blot with a We found that was phosphorylated by the FRAP but by an with the These the specificity of the We used the FRAP antibody to FRAP subcellular localization using We the of cells with this antibody in the or of FRAP antigenic A and was with the FRAP antibody A signal was also signal was by with the FRAP antigenic polypeptide In addition, signal was with the FRAP is predominantly in the In FRAP was on the of the that FRAP is localized to the plasma membrane. In addition, only a small of FRAP was found in the We have the FRAP antiserum with an FRAP antibody but in FRAP or Western The of FRAP shows to that of the ER and the Golgi apparatus. we performed with the FRAP antibody and an antibody for the ER or the Golgi apparatus in By we found that the FRAP with that of calnexin, a common ER marker protein and a common Golgi marker protein A recent suggests that FRAP is localized to Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). We examined the FRAP and in cells by the cells with the FRAP antibody and Mitotracker a Although there to be FRAP and their were that only a small of FRAP is localized in in cells this in for endogenous FRAP, we cells with a FRAP. with antibodies the that was with calnexin an C-terminal antibody of FRAP with calnexin These suggest that FRAP is localized to the ER and Golgi apparatus in we FRAP in other common mammalian cell lines with both and C-terminal FRAP We found FRAP in human and cancer cells that the ER/Golgi localization is cell localization is in cell FRAP localization in common mammalian cell FRAP localization was examined by with the N-terminal and C-terminal FRAP antibodies in human and B, FRAP in cells under conditions. cells were with rapamycin by for 1 sodium or FRAP was by with the N-terminal FRAP of cell signaling can occur by subcellular localization of components of the corresponding We FRAP in response to conditions that FRAP signaling. We found that FRAP in the or rapamycin conditions that FRAP signal transduction A was in cells that been from for an have been linked to FRAP sodium azide, an inhibitor of the been to phosphorylation Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, G. 2002; PubMed Scopus Google Scholar). treatment of cells with sodium the FRAP A recent that FRAP the and Chen J. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). treatment with B, an inhibitor of to FRAP in the in cells suggests that the is a FRAP localization we performed of cell After and of and membranes including and ER membranes were by FRAP was found in both the and The was further by to including the ER and Golgi apparatus membranes. the ER marker protein calnexin was found in the but in the We also found that FRAP was in the but the These are in with and suggest that FRAP is with the ER and the Golgi apparatus. FRAP may be with the or surface of the ER as a peripheral or membrane protein. it be localized the ER lumen. the of FRAP association with the ER, we the with conditions known to the membrane association of of A is used to peripheral membrane proteins membrane is in washing with the binding of the peripheral Golgi membrane protein Golgin-97 to membranes but on association of the membrane protein calnexin with ER/Golgi membranes only a of FRAP from the membranes with 4 but calnexin, from the membranes washing but FRAP from the membranes Finally, a pH pH Golgin-97 and FRAP from the although only calnexin These suggest that FRAP is an ER membrane protein that is tightly with the membrane. further the of FRAP ER we performed the protease We that FRAP were an membrane protein to the it be to protease the other an ER protein be from the ER is with We the with a of trypsin and chymotrypsin in the or of Triton is an membrane protein with the of its in the ER lumen. it amino acids to the with protease in the of calnexin was to a polypeptide with corresponding to its ER In the of calnexin was further In we found that FRAP was of Triton was or FRAP appears to be an ER protein. the that FRAP we the subcellular of by After of the and into was performed using the the ER/Golgi localization of the N terminus to amino and the FRAP the C terminus from amino FRAP localization further of amino acids amino from the C terminus to Golgi localization of FRAP and FRAP ER or Golgi localization with a further of amino acids from the C terminus and The two C-terminal and the and the These indicate that amino acids and amino acids are important for ER and Golgi respectively. In this we show that both endogenous and recombinant FRAP proteins are predominantly localized to the ER and Golgi apparatus in as by and cell A FRAP was with other common mammalian cell including human and that ER/Golgi localization is a common of FRAP and Houghton (3.Huang S. Houghton P. Curr. Opin. Investig. Drugs. 2002; 3: 295-304PubMed Google Scholar) localization of FRAP/mTOR in mammalian in the cell lines and we that the of FRAP ER/Golgi localization The the two studies the specificity of antibodies For example, antibody may only a of FRAP proteins of the of the corresponding is by the that the C-terminal FRAP antibody show more in and cells are also in with the of signaling been found that FRAP and the yeast proteins, and their as and are to membranes K. Maruki Y. Long X. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1448) Google Scholar, 14.Kim D. Sarbassov d.D. Ali S. King J. Latek R. Erdjument-Bromage H. Tempst P. Sabatini D. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2371) Google Scholar, Sarbassov d.D. Ali S. Latek R. K. Erdjument-Bromage H. Tempst P. Sabatini D. Mol. Cell. 2003; 11: Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. J. T. Mol. Biol. Cell. 2003; PubMed Scopus Google Scholar). that the yeast protein is with at an that appears to be the ER in yeast A. S. J. T. Mol. Biol. Cell. 2003; PubMed Scopus Google Scholar). The and is to K. Maruki Y. Long X. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1448) Google Scholar, 14.Kim D. Sarbassov d.D. Ali S. King J. Latek R. Erdjument-Bromage H. Tempst P. Sabatini D. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2371) Google Scholar, Sarbassov d.D. Ali S. Latek R. K. Erdjument-Bromage H. Tempst P. Sabatini D. Mol. Cell. 2003; 11: Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. J. T. Mol. Biol. Cell. 2003; PubMed Scopus Google that the ER membrane is important in the of the signaling indicate that the ER localization of is an conserved and is to an important role in further indicate that FRAP is with the surface of the ER treatment of the to of FRAP by the peripheral Golgi protein FRAP is to and to 4 or that FRAP is more tightly with the ER membrane than a peripheral membrane protein. FRAP may be or an membrane protein with the of its to the We have a to the important for FRAP ER/Golgi indicate that amino acids are critical for ER targeting FRAP this were to to the ER the other amino acids are essential for localization to the Golgi apparatus two to be with the the for ER targeting becomes localized to the Golgi apparatus The that FRAP is predominantly localized at the ER or Golgi is in of the that the sterol-SREBP signaling pathway is anchored at the ER and the Golgi apparatus as (25.Pahl H.L. Physiol. Rev. 1999; 79: 683-701Crossref PubMed Scopus (307) Google Scholar, M. J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Both the FRAP and SREBP pathways the of nutrients, amino acids and respectively a to the sterol signaling FRAP may on the ER/Golgi to amino and amino FRAP signaling is to be by with and also to other subcellular as the in response to signaling Like the that both the ER and the Golgi apparatus for regulation of sterol FRAP may also a localization organelles which is important for its signaling functions. of the important for ER/Golgi localization in cytoplasmic as as localization to the the ER and the Golgi apparatus may be used to the of FRAP proteins to the and which may be important for diverse as translation and ribosome We for with cell and the of the Zheng for