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Dispatches 3. Gann, A. (2010). Jacob and Monod: from operons to EvoDevo. Curr. Biol. 20, R718–R723. 4. Carroll, S.B. (2005). Evolution at two levels: on genes and form. PLoS Biol. 3, e245. 5. Hoekstra, H.E., and Coyne, J.A. (2007). The locus of evolution: evo devo and the genetics of adaptation. Evolution 61, 995–1016. 6. Wray, G.A. (2007). The evolutionary significance of cis-regulatory mutations. Nat. Rev. Genet. 8, 206–216. 7. Schmidt, D., Wilson, M.D., Ballester, B., Schwalie, P.C., Brown, G.D., Marshall, A., Kutter, C., Watt, S., Martinez-Jimenez, C.P., Mackay, S., et al. (2010). Five-vertebrate ChIP-seq reveals the evolutionary dynamics of transcription factor binding. Science 328, 1036–1040. 8. Tena, J.J., Gonza´lez-Aguilera, C., FernandezMin˜a´n, A., Va´zquez-Marı´n, J., Parra-Acero, H., Cross, J.W., Rigby, P.W.J., Carvajal, J.J., Wittbrodt, J., Go´mez-Skarmeta, J.L., et al. (2014). Comparative epigenomics in distantly related teleost species identifies conserved cis-regulatory nodes active during the vertebrate phylotypic period. Genome Res. 24, 1075–1085. 9. Kratochwil, C.F., and Meyer, A. (2015). Mapping active promoters by ChIP-seq profiling of H3K4me3 in cichlid fish - a first step to uncover cis-regulatory elements

in ecological model teleosts. Mol. Ecol. Resour. (in press). http://dx.doi.org/10.1111/ 1755-0998.12350. 10. Kratochwil, C.F., and Meyer, A. (2015). Closing the genotype-phenotype gap: Emerging technologies for evolutionary genetics in ecological model vertebrate systems. BioEssays 37, 213–226. 11. Villar, D., Berthelot, C., Aldridge, S., Rayner, T.F., Lukk, M., Pignatelli, M., Park, T.J., Deaville, R., Erichsen, J.T., Jasinska, A.J., et al. (2015). Enhancer evolution across 20 mammalian species. Cell 160, 554–566. 12. Brawand, D., Soumillon, M., Necsulea, A., Julien, P., Csa´rdi, G., Harrigan, P., Weier, M., Liechti, A., Aximu-Petri, A., Kircher, M., et al. (2011). The evolution of gene expression levels in mammalian organs. Nature 478, 343–348. 13. Villar, D., Flicek, P., and Odom, D.T. (2014). Evolution of transcription factor binding in metazoans - mechanisms and functional implications. Nat. Rev. Genet. 15, 221–233. 14. Guenther, C.A., Tasic, B., Luo, L., Bedell, M.A., and Kingsley, D.M. (2014). A molecular basis for classic blond hair color in Europeans. Nat. Genet. 46, 748–752. 15. Rubinstein, M., and de Souza, F.S.J. (2013). Evolution of transcriptional enhancers and

animal diversity. Philos. Trans. R. Soc. Lond. B Biol. Sci. 368, 20130017. 16. Abzhanov, A., Kuo, W.P., Hartmann, C., Grant, B.R., Grant, P.R., and Tabin, C.J. (2006). The calmodulin pathway and evolution of elongated beak morphology in Darwin’s finches. Nature 442, 563–567. 17. Attanasio, C., Nord, A.S., Zhu, Y., Blow, M.J., Li, Z., Liberton, D.K., Morrison, H., Plajzer-Frick, I., Holt, A., Hosseini, R., et al. (2013). Fine tuning of craniofacial morphology by distant-acting enhancers. Science 342, 1241006. 18. Rohner, N., Tschopp, P., and Tabin, C. (2014). Development: facial makeup enhancing our looks. Curr. Biol. 24 (1), R36–R38. 19. Rebeiz, M., Jikomes, N., Kassner, V.A., and Carroll, S.B. (2011). Evolutionary origin of a novel gene expression pattern through co-option of the latent activities of existing regulatory sequences. Proc. Natl. Acad. Sci. USA 108, 10036–10043. 20. Lynch, V.J., Nnamani, M.C., Kapusta, A., Brayer, K., Plaza, S.L., Mazur, E.C., Emera, D., Sheikh, S.Z., Gru¨tzner, F., Bauersachs, S., et al. (2015). Ancient transposable elements transformed the uterine regulatory landscape and transcriptome during the evolution of mammalian pregnancy. Cell Rep. (in press). http://dx.doi.org/10.1016/j.celrep. 2014.12.052.

Membrane Trafficking: Returning to the Fold(ER) Ana M. Perez-Linero and Manuel Mun˜iz* Departamento de Biologı´a Celular, Facultad de Biologı´a, Universidad de Sevilla, 41012 Sevilla, Spain Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocı´o/CSIC/Universidad de Sevilla, 41013 Sevilla, Spain *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cub.2015.02.007

Retrieval mechanisms are essential to dynamically maintain the composition and functional homeostasis of secretory organelles. A recent study has identified a novel class of cargo receptor that retrieves a specific subset of escaped ER folding machinery from the Golgi.

The endoplasmic reticulum (ER) is an amazing factory in charge of the synthesis of those luminal and membrane proteins that must be subsequently delivered by the secretory pathway to their proper functional destinations either at different organelles of the endomembrane system or outside of the cell. Newly synthesized secretory proteins are first inserted into the ER via the translocon, and subsequently a large battery of chaperones and enzymes carries out their

folding, assembly, and post-translational modifications, such as glycosylation or disulfide bond formation. Once correctly folded and assembled, secretory proteins are selectively incorporated as cargos into coat protein II (COPII)-coated vesicles, which transport them forward to the Golgi apparatus. This vesicular export flux is constantly challenging the protein composition and functional homeostasis of the ER. Although the ER resident proteins are not actively sorted into COPII

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vesicles, they still manage to escape from the ER. Indeed, they can passively enter the COPII vesicles, and thus exit the ER through bulk flow, being finally trafficked to the Golgi. Once there, the escaped proteins are captured and subsequently retrieved back to the ER in coat protein I (COPI)-coated retrograde transport vesicles. Active sorting of escaped ER proteins into COPI vesicles can be driven by direct interaction with the COPI coat or through

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Dispatches suggesting that this protein complex is involved in the ER retention of Gls1p. Furthermore, the authors find that Gls1p is a soluble protein that is still packaged, albeit very inefficiently and to a small extent, into COPII vesicles, indicating that Gls1p can exit the ER at a low rate by bulk flow and thus travel through the secretory pathway. These findings fit well with the idea that the Erv41p–Erv46p complex, which cycles between the ER and Golgi, contributes to ER retention by recognizing the escaped Gls1p at the Golgi lumen and subsequently retrieving it back to the ER. This possibility is strongly supported by further data obtained by the authors. First, both Erv41p and Erv46p physically interact in vitro and in vivo with Gls1p. Additionally, mutation of the dilysine COPI-binding motif in Erv46p mislocalizes Gls1p to the vacuole, indicating that the recycling from the Golgi to the ER of the Erv41p–Erv46p complex itself is required to dynamically localize Gls1p at the ER [5]. These results paint a picture of the Erv41p–Erv46p complex as a retrieval cargo receptor for escaped Gls1p and raised the question of whether there are other ER cargos for the Erv41p–Erv46p complex. Shibuya et al. [5] address this question by applying a whole cell stable isotope labeling by amino acids in culture (SILAC) proteomic analysis [11] to the erv41D and erv46D deletion mutant strains. This approach aided the authors in identifying other ER proteins that depended on the Erv41p–Erv46p complex for their ER localization, such as the peptidyl-prolyl isomerase Fpr2p [5]. Importantly, none of these potential cargos contain the HDEL motif typically recognized by the KDEL receptor, suggesting that the Erv41p–Erv46p complex functions as a retrieval receptor for a new class of ER resident proteins. These soluble cargos must therefore be recognized by the luminal domains of Erv46p or Erv41p, which have been recently proposed to promote protein–protein interactions [12]. Additional high-resolution structural studies of the luminal domain bound to cargo should provide key insight into the molecular mechanism by which the Erv41p–Erv46p receptor complex interacts with its ligands. Likewise, further investigation will be required to define the sorting signal present in

Golgi Decreasing pH

transmembrane cargo receptors that link the cargo with the coat [1]. Only two of these retrieval cargo receptors have been identified to date: the KDEL receptor, which retrieves soluble ER proteins bearing the carboxy-terminal KDEL motif (HDEL in yeast); and Rer1, which retrieves certain ER transmembrane proteins [2–4]. However, these two receptors and their known substrate range cannot account for the broad spectrum of ER resident proteins. In a recent issue of the Journal of Cell Biology, the Barlowe lab [5] identifies a new class of retrieval cargo receptor for a specific subset of ER resident proteins in yeast and, most importantly, describes its operating mechanism. A pioneering proteomic analysis of in vitro generated COPII vesicles previously performed by the Barlowe lab led to the identification, among others, of two novel related transmembrane proteins, Erv41p and Erv46p, as major passengers of the COPII vesicles in yeast [6]. Further characterization showed that these abundant and conserved proteins form a heteromeric complex that continuously cycles between the ER and Golgi via anterograde COPII and retrograde COPI vesicles. This efficient bidirectional trafficking relies on the presence in both proteins of COPII sorting motifs at their carboxyl termini, and an additional conserved COPI-binding dilysine motif at the carboxyl terminus of Erv46p [7]. However, despite the abundance of the Erv41p–Erv46p complex, its precise biological function in the early secretory pathway was elusive. An important clue came from the previous observation by the Otte lab that the lack of the Erv41p–Erv46p complex leads to the defective functioning of the ER resident glucosidase I (Gls1p) [8]. Gls1p cleaves the terminal glucose from the newly attached N-linked core glycan of glycoproteins in the ER and is known to function in the folding and quality control of newly synthesized glycoproteins [9,10]. From this finding, Shibuya et al. [5] reason that depletion of the Erv41p–Erv46p complex might somehow decrease the level of Gls1p in the ER and thus cause the defect in glucose trimming. Now, the authors show that Gls1p is indeed mislocalized to the vacuole and the extracellular medium in cells lacking the Erv41p–Erv46p complex,

COPI coat COPII coat

Erv41p–Erv46p Gls1p ER Current Biology

Figure 1. Model for the role of the Erv41p– Erv46p complex as a retrieval cargo receptor. The Erv41p–Erv46p complex binds to ‘escaped’ Gls1p in the reduced pH environment of the early Golgi. Then, it returns back to the ER in COPI vesicles and releases Gls1p in the near-neutral pH of the ER lumen. The empty Erv41p–Erv46p receptor is efficiently transported to the Golgi in COPII vesicles to initiate a new round of cargo retrieval. This model is based on the study from Shibuya et al. [5].

the ER resident proteins recognized by the Erv41p–Erv46p complex. Once identified, it will be also interesting to know whether there are toxins that use the Erv41p–Erv46p retrieval pathway to be targeted to the ER in an analogous manner to the mechanism exploited by KDEL-bearing toxins [13]. How does the Erv41p–Erv46p receptor work in retrograde transport? Organelles of the early secretory pathway exhibit a gradient of decreasing pH that begins at near neutrality at the ER and becomes more acidic in the Golgi [14]. Shibuya et al. [5] find that the Erv41p–Erv46p complex only binds to Gls1p under acidic pH conditions that mirror the reduced pH found in the lumen of the Golgi. Moreover, the increase of the pH in Golgi compartments by treatment with bafilomycin A1, an inhibitor of vacuolar ATPases [15], led to mislocalization of Gls1p. The data lead to a model (Figure 1) in which the escaped Gls1p is captured by the Erv41p–Erv46p complex in the reduced pH environment of early Golgi compartments for retrograde transport and is finally released in the neutral pH of the ER lumen. An analogous pH-dependent mechanism was proposed to operate for the KDEL receptor [16]. Importantly, the study by Shibuya et al. [5] highlights the essential role of the pH gradient for cargo sorting in retrograde

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Dispatches transport from the Golgi to the ER. However, the precise mechanism for cargo release at the ER still needs to be studied in more detail. Indeed, the authors show that a neutral pH is not sufficient to separate the cargo from the receptor, which suggests that additional unknown factors must be also involved in this process [5]. In addition, other interesting aspects remain to be addressed regarding the retrieval role of the Erv41p–Erv46p complex, such as whether its retrograde trafficking is constitutive or regulated by cargo recognition, as seen with the KDEL receptor [17]. In addition to its role as a retrieval receptor, the Erv41p–Erv46p complex has recently been assigned another transport function. A new study by the Yoda lab [18] reports that the Erv41p–Erv46p complex plays a role as an anterograde cargo receptor for the Golgi mannosyltransferase Ktr4p during ER export. Anterograde cargo receptors link newly synthesized secretory proteins to the COPII coat to facilitate their uptake into COPII vesicles for efficient transport to the Golgi. Like the retrieval receptors, they are also continuously cycling between the ER and Golgi and their receptor function has also been proposed to be regulated in a pH-dependent manner [1]. Further investigation will be required to understand how anterograde and retrograde cargo receptor activities can be coordinated to dynamically maintain the composition and functional homeostasis of the early secretory pathway.

Erv41p-Erv46p complex serves as a retrograde receptor to retrieve escaped ER proteins. J. Cell Biol. 208, 197–209. 6. Otte, S., Belden, W.J., Heidtman, M., Liu, J., Jensen, O.N., and Barlowe, C. (2001). Erv41p and Erv46p: new components of COPII vesicles involved in transport between the ER and Golgi complex. J. Cell Biol. 152, 503–518. 7. Otte, S., and Barlowe, C. (2002). The Erv41p-Erv46p complex: multiple export signals are required in trans for COPII-dependent transport from the ER. EMBO J. 21, 6095–6104. 8. Welsh, L.M., Tong, A.H., Boone, C., Jensen, O.N., and Otte, S. (2006). Genetic and molecular interactions of the Erv41p-Erv46p complex involved in transport between the endoplasmic reticulum and Golgi complex. J. Cell Sci. 119, 4730–4740. 9. Moremen, K.W., Trimble, R.B., and Herscovics, A. (1994). Glycosidases of the asparagine-linked oligosaccharide processing pathway. Glycobiology 4, 113–125. 10. Hitt, R., and Wolf, D.H. (2004). DER7, encoding alpha-glucosidase I is essential for degradation of malfolded glycoproteins of the endoplasmic reticulum. FEMS Yeast Res. 4, 815–820. 11. Frohlich, F., Christiano, R., and Walther, T.C. (2013). Native SILAC: metabolic labeling of proteins in prototroph microorganisms based on lysine synthesis regulation. Mol. Cell. Proteomics 12, 1995–2005.

12. Biterova, E.I., Svard, M., Possner, D.D., and Guy, J.E. (2013). The crystal structure of the lumenal domain of Erv41p, a protein involved in transport between the endoplasmic reticulum and Golgi apparatus. J. Mol. Biol. 425, 2208–2218. 13. Sandvig, K., and van Deurs, B. (2002). Membrane traffic exploited by protein toxins. Annu. Rev. Cell. Dev. Biol. 18, 1–24. 14. Paroutis, P., Touret, N., and Grinstein, S. (2004). The pH of the secretory pathway: measurement, determinants, and regulation. Physiology 19, 207–215. 15. Llopis, J., McCaffery, J.M., Miyawaki, A., Farquhar, M.G., and Tsien, R.Y. (1998). Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc. Natl. Acad. Sci. USA 95, 6803–6808. 16. Scheel, A.A., and Pelham, H.R. (1996). Purification and characterization of the human KDEL receptor. Biochemistry 35, 10203–10209. 17. Aoe, T., Lee, A.J., van Donselaar, E., Peters, P.J., and Hsu, V.W. (1998). Modulation of intracellular transport by transported proteins: insight from regulation of COPI-mediated transport. Proc. Natl. Acad. Sci. USA 95, 1624–1629. 18. Noda, Y., Hara, T., Ishii, M., and Yoda, K. (2014). Distinct adaptor proteins assist exit of Kre2-family proteins from the yeast ER. Biol. Open 3, 209–224.

Enhancers: Holding Out for the Right Promoter David S. Lorberbaum and Scott Barolo*

REFERENCES 1. Dancourt, J., and Barlowe, C. (2010). Protein sorting receptors in the early secretory pathway. Annu. Rev. Biochem. 79, 777–802. 2. Munro, S., and Pelham, H.R. (1987). A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, 899–907. 3. Semenza, J.C., Hardwick, K.G., Dean, N., and Pelham, H.R. (1990). ERD2, a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway. Cell 61, 1349–1357. 4. Sato, K., Sato, M., and Nakano, A. (1997). Rer1p as common machinery for the endoplasmic reticulum localization of membrane proteins. Proc. Natl. Acad. Sci. USA 94, 9693–9698. 5. Shibuya, A., Margulis, N., Christiano, R., Walther, T.C., and Barlowe, C. (2015). The

Department of Cell and Developmental Biology and Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cub.2015.01.039

Some transcriptional enhancers work best with one type of promoter, while ignoring others. How widespread is such specificity across the genome? A new study finds that, in a fair fight, most enhancers prefer to activate promoters resembling those of their parent genes.

To developmental biologists, there are two kinds of genes. Most of our time is spent thinking about what we might call the ‘interesting’ category, which covers all genes whose expression is differentially regulated — that is, genes that are more strongly expressed in cell A compared to

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cell B, or at stage X compared to stage Y. Differential gene expression, after all, is the cornerstone of development and many other complex biological processes: it’s what makes a cell different from its neighbor. The second category — genes whose expression is consistent

Membrane trafficking: returning to the fold(ER).

Retrieval mechanisms are essential to dynamically maintain the composition and functional homeostasis of secretory organelles. A recent study has iden...
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