theguaninenucleotideexchangefactorgea1 rescuesanisoform...
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DOI: 10.19185/matters.201609000004 Matters | 1 of 7
DisciplinesCell Biology
KeywordsGlucose StarvationGuanineNucleotide ExchangeFactor14-3-3 ProteinVesicular TrafficGlycogen Metabolism
Type of ObservationStandalone
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Submitted Sep 5th, 2016 Published Nov 2nd, 2016
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The guanine nucleotide exchange factor Gea1rescues an isoform-specific 14-3-3 phenotypeAnne Clancy, Bianca Schrul, Blanche SchwappachMolecular Biology, Universitätsmedizin Göttingen,; Molecular Biology, Universitätsmedizin Göttingen (DE)
14-3-3 proteins are abundant modulators of cellular processes, in particular signal transduction.They function by binding to a broad spectrum of client proteins, thus affecting client proteinlocalisation or function(Gardino 2011[1])(Morrison 2009[2]). Animals and plants express 14-3-3 proteins encoded by several genes, which has made it difficult to study their unique ratherthan shared functions. The yeast Saccharomyces cerevisiae possesses only two highly homolo-gous 14-3-3 genes, BMH1 and BMH2. Using this model system we now uncover novel aspectsof functional specificity between the two yeast 14-3-3s. We show that bmh1 but not bmh2 cellsdisplay an altered morphology of the endomembrane system and specific trafficking defectsunder glucose starvation. This but not a second phenotype specific to the bmh1 strain, i.e. theaccumulation of glycogen, was rescued by overexpression of the nucleotide exchange factorGea1, suggesting a role for Bmh1 in Gea1’s function or regulation.
ObjectiveHere, we aimed at dissecting specific cellular roles of Bmh1 using the YFP reporter assayand morphological analysis by transmission electron microscopy.
IntroductionSubstantial progress has been made in understanding the interaction of 14-3-3 proteinswith individual client proteins at a molecular level and in elucidating the functional con-sequences of individual interactions(Obsil 2011[4] )(Oecking 2009[5] ). However, in manyexperiments addressing the cellular roles of 14-3-3, they are treated as a generic entity al-though they are always present as a complexmixture of different homo- and heterodimers,and individual isoforms have been shown to bind with different affinities to a single sub-strate(Kilisch 2016[6] ). Since mammals have seven different isoforms and plants havethirteen, it has been challenging to study their functional divergence in these organisms,and our understanding of the unique functions of the different 14-3-3 isoforms is lim-ited. This problem is beginning to be tackled in different model organisms - Xenopuslaevis(Lau 2006[7] ) and Arabdopsis thaliana(Paul 2012[8] ) - and several instances of 14-3-3-specific interactions with well-studied clients such as the plant plasma membraneproton pump(Pallucca 2014[9] ) or the ERK1/2 scaffold KSR1(Jagemann 2008[10] ) havebeen described andmechanistically dissected. The genome of Saccharomyces cerevisiae en-codes only two highly homologous 14-3-3 proteins, Bmh1 and Bmh2 (93% sequence iden-tity), and either deletion strain is viable. In most laboratory strains the double dele-tion strain is not viable, illustrating the existence of overlapping functions of Bmh1 andBmh2 and consistent with a largely overlapping interactome of the yeast 14-3-3s(Panni2010[11] ). However, distinct phenotypes have been described in the individual deletionstrains(Gelperin 1995[12] )(Michelsen 2006[13] )(Trembley 2014[14] ). We set out to useyeast as a simple model system to further dissect isoform-specific 14-3-3 functions. Ithas been shown that a yeast multimeric membrane reporter protein that exposes argi-nine (R)-based COPI binding motifs (Pmp2-cc-YFP-LRKRS, from now on referred to asYFP reporter) is mislocalised specifically in bmh1 deletion cells(Michelsen 2006[13]). Im-portantly, overexpression of BMH2 in a bmh1 strain did not rescue the specific traffickingphenotype of the bmh1 strain, consistent with the notion that the phenotype is not merelydue to changes in 14-3-3 protein abundance in the cell, but that it indeed represents func-tional specificity between the two isoforms.
The guanine nucleotide exchange factor Gea1 rescues an isoform-specific 14-3-3 phenotype
DOI: 10.19185/matters.201609000004 Matters | 2 of 7
The guanine nucleotide exchange factor Gea1 rescues an isoform-specific 14-3-3 phenotype
DOI: 10.19185/matters.201609000004 Matters | 3 of 7
Gea1 rescues bmh1-specific phenotypes, that is, alterations in protein trafficking along the secretory path-way and endomembrane morphology.(a) Localisation of a YFP reporter (Pmp2-cc-YFP-LRKRS) in log-phase cultures before and 90min after glucosewithdrawal(plus D, minus D) in the indicated strains. FM4-64(Vida 1995[3] ) labels vacuolar (vac) membranes, to assign stainingpatterns to either the vacuole or the perinuclear ER for quantification. Scale bar 5 μm.(b) Quantification of subcellular YFP-reporter localisation as in (a). Cells showing predominantly ER, equal ER andvacuole, or predominantly vacuole staining were counted. Between 422 and 491 cells were counted from 6 biologicalreplicates. Error bars are shown only in the negative direction and are standard error of the mean (S.E.M), p values werecalculated using a two-tailed Student’s test.(c-h) bmh1 shows reduced vesicular traffic and increased glycogen accumulation under glucose starvation. Represen-tative electron micrographs of (c) wild type cells showing multivesicular bodies, (e) bmh2 showing vesicle clusters,(g) bmh1 showing glycogen accumulation. Scale bars, black=1,000 nm, white=500 nm. CW: cell wall, ER: endoplasmicreticulum, MVB: multivesicular body, VC: vesicle cluster, LD: lipid droplet, G: glycogen, V: vacuole. For quantification,electron micrographs of each strain were examined and several morphological features were quantified: (d) MVBs, (f)VC (>3 vesicles) and (h) glycogen accumulation. Wild type n=391, bmh1 n=342, bmh2 n=361. *** indicates p<0.001, *indicates p<0.005 as calculated using the chi-squared test.(i) Photograph of the indicated strains spotted onto SC plates and exposed to iodine vapour reveals increased glycogencontent in bmh1 strain.(j, k, l)Quantification of electron micrographs obtained as in (c), from bmh1 strains expressing GEA1 and bmh1 and wildtype strains transformed with an empty vector as indicated, (j) MVBs, (k) VC (>3 vesicles), (l) glycogen accumulation.wt + empty vector n=405, bmh1 + empty vector n= 409, bmh1 + GEA1 n=401. *** indicates p<0.001, * indicates p<0.005as calculated using the chi-squared test.(m) Localisation of YFP reporter in log phase cultures expressing HA-GEA1 from the strong PGK promoter in eitherwt, bmh1 or bmh2 cells. FM4-64 marks vacuole membranes.
The guanine nucleotide exchange factor Gea1 rescues an isoform-specific 14-3-3 phenotype
DOI: 10.19185/matters.201609000004 Matters | 4 of 7
Results and discussionBmh1-specific function in protein transport along the secretory pathwayTo better understand the specific role of Bmh1 in membrane transport of the YFP reporter we assessed this phenomenonquantitatively by determining the subcellular localisation of the reporter in wild type, bmh1 and bmh2 knockout strains(Fig. 1a, b). In logarithmic phase, the reporter was predominately localised to the vacuole of wild type and bmh2 cells(left panels (a)) in ca. 80% of cells, quantification in (b). In contrast, the reporter localised to the vacuole of only 30%and was visible in the ER of 65% of bmh1 cells, suggesting that trafficking of our YFP reporter along the secretorypathways was compromised. These phenotypes became even more pronounced during stationary phase(Michelsen2006[13] ). Since stationary yeast cultures typically experience glucose deprivation we assayed whether this was therelevant parameter determining the Bmh1 requirement for reporter localisation. Indeed, upon glucose starvation, therequirement for Bmh1 was even more pronounced and the reporter only fully reached the vacuole in 10% of cells, asopposed to 50% in the wild type and bmh2 deletion strains (Fig. 1a, b). We concluded that the role of the 14-3-3 Bmh1in targeting the YFP reporter to the vacuole is particularly relevant under conditions of glucose starvation.Bmh1 but not Bmh2 is required to maintain the global morphology of the endomembrane system.To assess the global morphology of the endomembrane system, we investigated the wild type, bmh1 and bmh2 dele-tion strains after glucose starvation using transmission electron microscopy (TEM) (Fig. 1c, e, g). Qualitative assess-ment revealed three different subcellular structures whose abundance varied with the genotypes, that is, multivesicularbodies (MVBs, Fig. 1c), vesicle clusters (VC, Fig. 1e) and glycogen granules (Fig. 1g). Quantification revealed thatthe bmh1 strain possessed less MVBs (Fig. 1d) and less VCs (Fig. 1f), consistent with global alterations in vesiculartraffic. Furthermore, we observed substantial accumulation of glycogen granules in the bmh1 strain (Fig. 1h), whichwas independently confirmed by iodine staining (Fig. 1i).The guanine nucleotide exchange factor Gea1 rescues Bmh1-specific alterations in the endomembrane system/vesicular trans-port but not glycogen accumulationGuanine nucleotide exchange factor (GEF) proteins activate the GTPase Arf1 by conversion of GDP-Arf1 to active GTP-Arf1. In consequence, the N-terminal myristoylation moiety is exposed and is able to anchor Arf1 in the Golgi mem-brane, where it recruits COPI coat components and initiates the process of COPI budding(Liu 2009[15] ). Gea1 is oneof four GEFs found in yeast(Peyroche 1996[16] ). Together with mammalian GBF1, Gea1 and Gea2 are part of a sub-family of GEFs distinct from the Sec7 subfamily. Different functionalities of the subfamilies are highlighted by the factthat GEA1 cannot suppress the temperature sensitivity of a sec7 mutant and vice versa. Phosphorylation of GBF1 wasshown to have a critical role in Golgi disassembly(Miyamoto 2007[17] ) and gea1/2 mutants showed disrupted Golgi inyeast(Peyroche 2001[18] ), supporting the notion that they have similar roles in the two systems. Furthermore, Gea1 hasbeen shown to increase the rate of GTPɣS binding to Arf1 in vitro(Peyroche 1996[16]) suggesting that it may affectCOPI-dependent processes in vivo.
In mammalian cells, AMPK kinase is known to phosphorylate the GEF GBF1(Miyamoto 2007[17])(Claude 1999[19] ).It is thought that this regulation attenuates the function of GBF1 when the intracellular concentration of ATP drops.Therefore, yeast AMPK may regulate Gea1 function in response to glucose availability. Hence, we tested whether wecould suppress Bmh1-specific phenotypes by overexpression of GEA1, to date the better studied of the two yeast ho-mologues of GBF1(Claude 1999[19]), to supply the cell with additional GEF activity and thus counteract a putativedown-regulation of GEF function in the bmh1 deletion strain (Fig. 1j, k, l, m). Indeed, overexpression of GEA1 restoredthe localisation of the YFP reporter to the vacuole in the bmh1 strain (Fig. 1m), just as observed for the wild typeand bmh2 strains. Compare Fig. 1a right, without GEA1 overexpression and Fig. 1m showing a cell in which GEA1was overexpressed. Similarly, GEA1 overexpression in the bmh1 strain increased the abundance of MVBs (Fig. 1j) andVCs (Fig. 1k) to levels even higher than in the wild type strain. In contrast, the accumulation of glycogen granules inthe bmh1 background was unaffected by overexpression of the Arf-GEF GEA1 (Fig. 1l). These results are consistent withthe notion that the global alterations in vesicular traffic observed in the bmh1 strain (reduction of reporter trafficking tothe vacuole, reduction inMVBs and VCs) are downstream of Gea1 function and that Gea1 is less functional in the absenceof this 14-3-3 protein. However, overexpression ofGEA1 did not suppress glycogen accumulation in the bmh1 strain.This indicates that Bmh1 action is upstream of Gea1 in a pathway that culminates in changes in vesicular trafficking,yet Bmh1 function is independent of Gea1 in the pathway leading to glycogen accumulation.
ConclusionsGEA1 overexpression rescues the isoform-specific protein trafficking defect seen in bmh1 strains, suggesting that Bmh1has a regulatory effect on the activity of Gea1. Gea1 did not abrogate the glycogen build-up in bmh1 cells, indicatingthat Bmh1 has additional effects on pathways distinct from the protein-trafficking processes involving Gea1.
ConjecturesIt would be interesting to test whether the effect of Bmh1 on Gea1 is direct or via some upstream regulatory factor and to
The guanine nucleotide exchange factor Gea1 rescues an isoform-specific 14-3-3 phenotype
DOI: 10.19185/matters.201609000004 Matters | 5 of 7
identify this factor. This could then delineate the various pathways specifically affected by Bmh1 and enable elucidationof the underlying molecular mechnisms.
AlthoughGEA1 andGEA2 are redundant in some functions, and the double deletion is inviable (Peyroche 1996[16] ),the genes have only 51% sequence identity, and independent functions have been demonstrated. Spang et al. showedthat GEA1 and GEA2 differentially rescue COPI mutant strains, and that the corresponding mutants have differingsynthetic lethality with arf1 (Spang 2001[20] ). Only gea2 is lethal in combination with arf1, indicating it has rolesother than Arf1 activation. It has been linked to Arf3 for example as having a role in actin organisation (Zakrzewska2003[21] ). It remains unknown whether GEA2 would also rescue the Bmh1-specific phenotypes seen here; if not it wouldfurther demonstrate the differential specificity of the GEFs, and beg the question whether or not both Gea1 and Gea2are regulated in a Bmh1-mediated manner.Snf1, the yeast AMPK kinase, and Reg1, a regulatory subunit of the phosphatase Glc7, which is known to regulate Snf1and may also act on Snf1 substrates (Ludin 1998[22] ), may provide a further link between Bmh1 and Gea1. Reg1 isone of the best-characterised binding partners of Bmh1 (Panni 2010[11] ) (Dombek 2004[23] ) (Mayordomo 2003[24] ).The reg1 deletion strain also accumulates glycogen (Huang 1996[25] ), similar to the bmh1 strain. Snf1’s main functionis to adapt the cell to changes in glucose availability (Gancedo 1998[26] ) (Thevelein 1994[27] ) (Wilson 1996[28] ). Itis tempting to propose that Bmh1 may impact on a phosphorylation-controlled activation mechanism targeting Gea1,thus providing another layer of regulation of Gea1’s GEF activity. The first step in testing this hypothesis would be todemonstrate differential phosphorylation of endogenous Gea1 in different bmh deletion strains.
Additional Information
Methods and supplementary materialPlease see https://sciencematters.io/articles/201609000004.
Funding statementThis work was funded by the DFG in the framework of SFB1190 [Project Z03 to B.S.] and the University Medical Center,Göttingen (UMG).
AcknowledgementsWe thank D. Riedel, D. Wenzel and G. Heim for technical advice regarding electron microscopy. We are grateful to EricArakel, Anne Spang, and Maya Schuldiner for advice and very helpful discussions throughout the project.
Ethics statementNot applicable.
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