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Apostolos Argiropoulos, Emmanuel Spanakis, Sophia Rhizopoulou (2017) Functional micromorphology of petals of Chaenomeles japonica exposed to humid and cold season. Acta Physiologia Plantarum 39:246 DOI 10.1007/s11738-017-2542-2

Abstract: In this study, micro- and nano-traits of petal epidermises of flowers of Chaenomeles japonica extended under environmental conditions, during the humid and cold period of the year, are presented. The outer (abaxial) and the inner (adaxial) epidermises of petals of C. japonica consist of convex and papillae cells, respectively, that are covered by epicuticular wrinkled relief further ornamented by submicron motifs, forming interfaces between floral tissues and environment. Structural epidermal features of the petal relief at the nanoscale level reveal different functionality on the two sides of the corolla. The cuticular folds of convex epidermal cells display declining water retention on the outer petal surface and the exposed side of the corolla to the environmental conditions. The cuticular folds of papillae epidermal cells increase in size the inner petal surface, in comparison with the outer surface; such traits facilitate light absorption and enhanced the contact area among folds and curvatures at the inner side of the corolla. It appears that nanometric surface structures of petals may be important adaptive features of C. japonica flowers, contributing to their performance in the field.

Balaska S, Myrianthopoulos V, Tselika M, Hatzinikolaou DG, Mikros E, Diallinas G. (2017) NmeA, a novel efflux transporter specific for nucleobases and nucleosides, contributes to metal resistance in Aspergillus nidulans. Mol Microbiol. 2017 Aug;105(3):426-439. doi: 10.1111/mmi.13708. Epub 2017 May 30. PubMed PMID:28509393.

Abstract: Through Minos transposon mutagenesis we obtained A. nidulans mutants resistant to 5-fluorouracil due to insertions into the upstream region of the uncharacterized gene nmeA, encoding a Major Facilitator Superfamily (MFS) transporter. Minos transpositions increased nmeA transcription, which is otherwise extremely low under all conditions tested. To dissect the function of NmeA we used strains overexpressing or genetically lacking the nmeA gene. Strains overexpressing NmeA are resistant to toxic purine analogues, but also, to cadmium, zinc and borate, whereas an isogenic nmeAΔ null mutant exhibits increased sensitivity to these compounds. We provide direct evidence that nmeA overexpression leads to efflux of adenine, xanthine, uric acid and allantoin, the latter two being intermediate metabolites of purine catabolism that are toxic when accumulated cytoplasmically due to relevant genetic lesions. By using a functional GFP-tagged version we show that NmeA is a plasma membrane transporter. Homology modeling and docking approaches identified a single purine binding site and a tentative substrate translocation trajectory in NmeA. Orthologues of NmeA are present in all Aspergilli and other Eurotiomycetes, but are absent from other fungi or non-fungal organisms. NmeA is thus the founding member of a new class of transporters essential for fungal success under specific toxic conditions.
Borbolis F, Flessa CM, Roumelioti F, Diallinas G, Stravopodis DJ, Syntichaki P. (2017) Neuronal function of the mRNA decapping complex determines survival of Caenorhabditis elegans at high temperature through temporal regulation of heterochronic gene expression. Open Biol. 2017 Mar;7(3). pii: 160313. doi: 10.1098/rsob.160313.
Abstract: In response to adverse environmental cues, Caenorhabditis elegans larvae can temporarily arrest development at the second moult and form dauers, a diapause stage that allows for long-term survival. This process is largely regulated by certain evolutionarily conserved signal transduction pathways, but it is also affected by miRNA-mediated post-transcriptional control of gene expression. The 5'-3' mRNA decay mechanism contributes to miRNA-mediated silencing of target mRNAs in many organisms but how it affects developmental decisions during normal or stress conditions is largely unknown. Here, we show that loss of the mRNA decapping complex activity acting in the 5'-3' mRNA decay pathway inhibits dauer formation at the stressful high temperature of 27.5°C, and instead promotes early developmental arrest. Our genetic data suggest that this arrest phenotype correlates with dysregulation of heterochronic gene expression and an aberrant stabilization of lin-14 mRNA at early larval stages. Restoration of neuronal dcap-1 activity was sufficient to rescue growth phenotypes of dcap-1 mutants at both high and normal temperatures, implying the involvement of common developmental timing mechanisms. Our work unveils the crucial role of 5'-3' mRNA degradation in proper regulation of heterochronic gene expression programmes, which proved to be essential for survival under stressful conditions.

D. Chamorro, B. Luna, J.-M. Ourcival, A. Kavgacı, C. Sirca, F. Mouillot, M. Arianoutsou & J. M. Moreno. (2017) Germination sensitivity to water stress in four shrubby species across the Mediterranean Basin. Plant Biology 19, 23–31, doi:10.1111/plb.12450

Abstract: Mediterranean shrublands are generally water-limited and fire-driven ecosystems. Seed-based post-fire regeneration may be affected by varying rainfall patterns, depending on species sensitivity to germinate under water stress. In our study, we considered the germination response to water stress in four species from several sites across the Mediterranean Basin. Seeds of species with a hard coat (Cistus monspeliensis, C. salviifolius, Cistaceae, Calicotome villosa, Fabaceae) or soft coat (Erica arborea, Ericaceae), which were exposed or not to a heat shock and smoke (fire cues), were made to germinate under water stress. Final germination percentage, germination speed and viability of seeds were recorded. Germination was modelled using hydrotime analysis and correlated to the water balance characteristics of seed provenance. Water stress was found to decrease final germination in the three hard-seeded species, as well as reduce germination speed. Moreover, an interaction between fire cues and water stress was found, whereby fire cues increased sensitivity to water stress. Seed viability after germination under water stress also declined in two hard-seeded species. Conversely, E. arborea showed little sensitivity to water stress, independent of fire cues. Germination responses varied among populations of all species, and hydrotime parameters were not correlated to site water balance, except in E. arborea when not exposed to fire cues. In conclusion, the species studied differed in germination sensitivity to water stress; furthermore, fire cues increased this sensitivity in the three hard-seeded species, but not in E. arborea. Moreover, populations within species consistently differed among themselves, but these differences could only be related to the provenance locality in E. arborea in seeds not exposed to fire cues.

P.G. Dimitrakopoulos , S. Koukoulas , A. Galanidis , P. Delipetrou, D. Gounaridis, K. Touloumi, M. Arianoutsou. 2017. Factors shaping alien plant species richness spatial patterns across Natura 2000 Special Areas of Conservation of Greece. Science of the Total Environment, 601-602, 461-468, dx.doi.org/10.1016/j.scitotenv.2017.05.220

Abstract: This paper aims to determine the main factors that shape the spatial patterns of alien plant species occurrence across Natura 2000 Special Areas of Conservation (SACs) inGreece, and quantify their influence. A series of spatial analysis techniques for the development of a spatial database of the factors involved, followed by a boosted negative binomial Generalised Additive Model for location scale and shape, were implemented. Native plant species richness, topography and hydrography, human population density, and a spatial preference to the northernwestern sites are the key factors that explain the variation in the occurrence of alien plant species. Native plant species richness and human population density have a positive effect on alien plant species presence, while topography aspects, such as elevation and slope, and the distance from the hydrographical network a negative one. All factors are indirectly linked to propagule pressure emphasizing the importance of human activities for the efforts on managing protected areas.

N.M. Fyllas, A. Christopoulou, A. Galanidis, C.Z. Michelaki, C. Giannakopoulos, P.G. Dimitrakopoulos, M. Arianoutsou, & Gloor M. (2017) Predicting species dominance shifts across elevation gradients at mountain forests in Greece under a warmer and drier climate. Regional Environmental Change, DOI 10.1007/s10113-016-1093-1, published on line 21 January 2017

Abstract: The Mediterranean Basin is expected to face warmer and drier conditions in the future, following projected increases in temperature and declines in precipitation. The aim of this study is to explore how forests dominated by Abies borisii-regis, Abies cephalonica, Fagus sylvatica, Pinus nigra and Quercus frainetto will respond under such conditions. We combined an individual- based model (GREFOS), with a novel tree ring data set in order to constrain tree diameter growth and to account for inter- and intraspecific growth variability. We used wood density data to infer tree longevity, taking into account inter- and intraspecific variability. The model was applied at three 500-m-wide elevation gradients at Taygetos in Peloponnese, at Agrafa on Southern Pindos and at Valia Kalda on Northern Pindos in Greece. Simulations adequately represented species distribution and abundance across the elevation gradients under current climate. We subsequently used the model to estimate species and functional trait shifts under warmer and drier future conditions based on the IPCC A1B scenario. In all three sites, a retreat of less drought-tolerant species and an upward shift of more drought-tolerant species were simulated. These shifts were also associated with changes in two key functional traits, in particular maximum radial growth rate and wood density. Drought-tolerant species presented an increase in their average maximal growth and decrease in their average wood density, in contrast to less drought tolerant species.

N. M.Fyllas, A. Christopoulou, A. Galanidis, Ch. Z. Michelaki, P. G. Dimitrakopoulos, Peter Z.Fulé, M. Arianoutsou (2017). Tree growth-climate relationships in a forest-plot network on Mediterranean mountains. Science of The Total Environment, 598,  393-403

Abstract: In this study we analysed a novel tree-growth dataset, inferred from annual ring-width measurements, of 7 forest tree species from 12 mountain regions in Greece, in order to identify tree growth – climate relationships. The tree species of interest were: Abies cephalonica, Abies borisii-regis, Picea abies, Pinus nigra, Pinus sylvestris, Fagus sylvatica and Quercus frainetto growing across a gradient of climate conditions with mean annual temperature ranging from 5.7 to 12.6 °C and total annual precipitation from 500 to 950 mm. In total, 344 tree cores (one per tree) were analysed across a network of 20 study sites. We found that water availability during the summer period (May–August) was a strong predictor of interannual variation in tree growth for all study species. Across species and sites, annual tree growth was positively related to summer season precipitation (PSP). The responsiveness of annual growth to PSP was tightly related to species and site specific measurements of instantaneous photosynthetic water use efficiency (WUE), suggesting that the growth of species with efficient water use is more responsive to variations in precipitation during the dry months of the year. Our findings support the importance of water availability for the growth of mountainous Mediterranean tree species and highlight that future reductions in precipitation are likely to lead to reduced tree-growth under climate change conditions.
de Vries RP, Riley R, Wiebenga A, Aguilar-Osorio G, Amillis S, Uchima CA, Anderluh G, Asadollahi M, Askin M, Barry K, Battaglia E, Bayram Ö, Benocci T, Braus-Stromeyer SA, Caldana C, Cánovas D, Cerqueira GC, Chen F, Chen W, Choi C, Clum A, Dos Santos RA, Damásio AR, Diallinas G, Emri T, Fekete E, Flipphi M, Freyberg S, Gallo A, Gournas C, Habgood R, Hainaut M, Harispe ML, Henrissat B, Hildén KS, Hope R, Hossain A, Karabika E, Karaffa L, Karányi Z, Kraševec N, Kuo A, Kusch H, LaButti K, Lagendijk EL, Lapidus A, Levasseur A, Lindquist E, Lipzen A, Logrieco AF, MacCabe A, Mäkelä MR, Malavazi I, Melin P, Meyer V, Mielnichuk N, Miskei M, Molnár ÁP, Mulé G, Ngan CY, Orejas M, Orosz E, Ouedraogo JP, Overkamp KM, Park HS, Perrone G, Piumi F, Punt PJ, Ram AF, Ramón A, Rauscher S, Record E, Riaño-Pachón DM, Robert V, Röhrig J, Ruller R, Salamov A, Salih NS, Samson RA, Sándor E, Sanguinetti M, Schütze T, Sepčić K, Shelest E, Sherlock G, Sophianopoulou V, Squina FM, Sun H, Susca A, Todd RB, Tsang A, Unkles SE, van de Wiele N, van Rossen-Uffink D, Oliveira JV, Vesth TC, Visser J, Yu JH, Zhou M, Andersen MR, Archer DB, Baker SE, Benoit I, Brakhage AA, Braus GH, Fischer R, Frisvad JC, Goldman GH, Houbraken J, Oakley B, Pócsi I, Scazzocchio C, Seiboth B, vanKuyk PA, Wortman J, Dyer PS, Grigoriev IV (2017) Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus. Genome Biol. Feb 14;18(1):28. doi: 10.1186/s13059-017-1151-0
Abstract: BACKGROUND: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. RESULTS: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. CONCLUSIONS: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.
Diallinas G. (2017) Transceptors as a functional link of transporters and receptors Microbial Cell, Vol. 4, No. 3, pp. 69 - 73; DOI: 10.15698/mic2017.03.560
Abstract: Cells need to communicate with their environment in order to obtain nutrients, grow, divide and respond to signals related to adaptation in changing physiological conditions or stress. A very basic question in biology is how cells, especially of those organisms living in rapidly changing habitats, sense their environment. Apparently, this question is of particular importance to all free-living microorganisms. The critical role of receptors, transporters and channels, transmembrane proteins located in the plasma membrane of all types of cells, in signaling environmental changes is well established. A relative newcomer in environment sensing are the so called transceptors, membrane proteins that possess both solute transport and receptor-like signaling activities. Now, the transceptor concept is further enlarged to include micronutrient sensing via the iron and zinc high-affinity transporters of Saccharomyces cerevisiae. Interestingly, what seems to underline the transport and/or sensing function of receptors, transporters and transceptors is ligand-induced conformational alterations recognized by downstream intracellular effectors.
Martzoukou O, Amillis S, Zervakou A, Christoforidis S, Diallinas G. (2017) The AP-2 complex has a specialized clathrin-independent role in apical endocytosis and polar growth in fungi. Elife. 2017 Feb 21;6. pii: e20083. doi: 10.7554/eLife.20083. [Epub ahead of print]
Abstract: Filamentous fungi provide excellent systems for investigating the role of the AP-2 complex in polar growth. Using Aspergillus nidulans, we show that AP-2 has a clathrin-independent essential role in polarity maintenance and growth. This is in line with a sequence analysis showing that the AP-2 β subunit (β2) of higher fungi lacks a clathrin-binding domain, and experiments showing that AP-2 does not co-localize with clathrin. We provide genetic and cellular evidence that AP-2 interacts with endocytic markers SlaBEnd4 and SagAEnd3 and the lipid flippases DnfA and DnfB in the sub-apical collar region of hyphae. The role of AP-2 in the maintenance of proper apical membrane lipid and cell wall composition is further supported by its functional interaction with BasA (sphingolipid biosynthesis) and StoA (apical sterol-rich membrane domains), and its essentiality in polar deposition of chitin. Our findings support that the AP-2 complex of dikarya has acquired, in the course of evolution, a specialized clathrin-independent function necessary for fungal polar growth.
Meletiou-Christou MS, Rhizopoulou S (2017) Leaf functional traits of four evergreen species growing in Mediterranean environmental conditions. Acta Physiologiae Plantarum 39:34(1-13), doi:10.1007/s11738-016-2330-4
Abstract: In this study, physiological and biochemical responses of native and introduced evergreens to the Mediterranean region, co-occurring under ambient conditions, are presented. Carbon and nitrogen compounds have been investigated in fully expanded leaves of the Mediterranean evergreens Laurus nobilis and Nerium oleander and introduced to the Mediterranean region Asian evergreens Ligustrum japonicum and Pittosporum tobira in May (end of the growing season), July (middle of the drought season), October (beginning of the wet season), and January (middle of the cold season). The above-mentioned species are subjected to seasonal fluctuations of climatic stimuli, in the eastern Mediterranean region. During the most vigorous vegetative growth in spring, the species maintained elevated chlorophyll, soluble sugar, and starch content, at rising water potential and CO2 assimilation. Leaf proline content increased during unfavourable drought conditions, concomitantly with reduced leaf osmotic potential; in addition, declining water availability, during the dry season, had a significant impact on leaf water potential and turgor. Total lipid and nitrogen content increased during the wet and cold seasons. In general, lipid content was higher in mature leaves of the Mediterranean evergreens (L. nobilis and N. oleander) throughout the year, in comparison with that of the introduced evergreens (L. japonicum and P. tobira), while fatty acid composition seems to be species specific. Although differences in leaf attributes have been investigated between native and introduced species in the Mediterranean region, fully expanded leaves of the studied species did not appear to respond differently to the seasonality of the Mediterranean ecosystem.

Papadaki GF, Amillis S, Diallinas G. Substrate Specificity of the FurE Transporter Is Determined by Cytoplasmic Terminal Domain Interactions (2017) Genetics. 2017 Oct 4. pii: genetics.300327.2017. doi: 10.1534/genetics.117.300327. [Epub ahead of print] PubMed PMID: 28978674

Abstract: FurE, a member of the NCS1 transporter family in Aspergillus nidulans, is specific for allantoin, uric acid, uracil and related analogues. Herein, we show that C- or N-terminally truncated FurE transporters (FurE-ΔC or FurE-ΔΝ) present increased protein stability, but also inability for uric acid transport. To better understand the role of cytoplasmic terminal regions, we characterized genetic suppressors that restore FurE-ΔC-mediated uric acid transport. Suppressors map in the periphery of the substrate-binding site (Thr133 in TMS3 and Val343 in TMS8), an outward-facing gate (Ser296 in TMS7, Ile371 in TMS9, Tyr392 and Leu394 in TMS10) or in flexible loops (Asp26 in LN, Gly222 in L5, Asn308 in L7). Selected suppressors were shown to also restore the wild-type specificity of FurE-ΔΝ, suggesting that both C- and/or N-terminal domains are involved in intramolecular dynamics critical for substrate selection. A direct, substrate-sensitive, interaction of C- and/or N-terminal domains was supported by bimolecular fluorescence complementation assays. To our knowledge, this is the first case where not only the function, but also, the specificity of a eukaryotic transporter is regulated by its terminal cytoplasmic regions.
Radea C., Louvrou I., Bakolitsas K. & A. Economou-Amilli (2017) Local endemic and thretened freshwater hydrobiids of Western Greece: elucidation of anatomy and new records. Folia Malacologica 25. https://doi.org/1012657/formal.025.001
Abstract: Islamia trichoniana Radoman, Pseudoislamia balcanica Radoman and Trichonia trichonica Radoman are local endemics and threatened hydrobiids living in Lake Trichonis, Western Greece. The rough initial description of their reproductive organs (i.e. penis, bursa copulatrix and receptacula seminis) is supplemented with new data. The radulae of I. trichoniana and P. balcanica are described in detail for the first time. New records of the three species are given and preliminary data about their feeding regime are provided.

Rhizopoulou S. (2017) Fascinating landscapes of “Hypnerotomachia Poliphili” (1499): a source for research of plant diversity, horticulture and culture. Acta Horticulturae, 1189: 19−24


Abstract: "Hypnerotomachia Poliphili" (HP) is a literary source of semi-natural and human-induced landscapes that centred on seductive environmental attributes, such as gardens, groves, pergolas and dispersed ancient ruins, which represent knowledge derived from the tradition and experience of ages. HP is the short title of one of the most influential books of the Renaissance (the first edition was published by Aldus Manutius in Venice, in 1499); it is the story of a man (Poliphilos), who falls asleep and dreams of traversing landscapes and gardens containing plants, ancient ruins and allegorical figures, in order to be reunited with his beloved (Polia). Interest in this novel has been revived, due to substantial influence on landscape and garden design. In this study, attention has been given to the botanical section preserved in the text. Paying attention to narratives provides a source for (re)considering conceptions of landscapes, nature and culture, wild and domesticated plant life. Arboreal and bush plants here quoted disclose aspects of plant diversity and ecophysiology; among a large number of plant taxa, roses, myrtle, pines, cypresses, laurels, acanthus, oaks, olive trees, juniper, ivy, thyme, oregano, violets, wormwood and cyclamens were the most frequently cited. Also, the seasonality of Mediterranean landscapes, indicated by flowering and fruit-bearing indigenous plants, can be traced through textual passages. Its wood-engraved illustrations of portrayed landscapes and botanic sketches (e.g., ferns, palms, oaks, cypresses, box trees and foliage of laurel, acanthus, grapevine and ivy) constitute evidence for both plant diversity and natural history, at the end of the fifteenth-century.

Seebens H., Blackburn T.M., Dyer E.E., Genovesi P., Hulme P.E., Jeschke J.M., Pagad Sh., Pysek P., Winter M., Arianoutsou M., et al. (2017) No saturation in the accumulation of alien species worldwide. Nature Communications, 8, 14435 doi: 10.1038/ncomms14435.

Abstract: Although research on human-mediated exchanges of species has substantially intensified during the last centuries, we know surprisingly little about temporal dynamics of alien species accumulations across regions and taxa. Using a novel database of 45,813 first records of 16,926 established alien species, we show that the annual rate of first records worldwide has increased during the last 200 years, with 37% of all first records reported most recently (1970–2014). Inter-continental and inter-taxonomic variation can be largely attributed to the diaspora of European settlers in the nineteenth century and to the acceleration in trade in the twentieth century. For all taxonomic groups, the increase in numbers of alien species does not show any sign of saturation and most taxa even show increases in the rate of first records over time. This highlights that past efforts to mitigate invasions have not been effective enough to keep up with increasing globalization.

Sioupouli G, Lambrinidis G, Mikros E, Amillis S, Diallinas G. (2017) Cryptic purine transporters in Aspergillus nidulans reveal the role of specific residues in the evolution of specificity in the NCS1 family. Mol Microbiol. 2017 Jan;103(2):319-332. doi: 10.1111/mmi.13559. Epub 2016 Nov 25.
Abstract: NCS1 proteins are H(+) or Na(+) symporters responsible for the uptake of purines, pyrimidines or related metabolites in bacteria, fungi and some plants. Fungal NCS1 are classified into two evolutionary and structurally distinct subfamilies,  known as Fur- and Fcy-like transporters. These subfamilies have expanded and functionally diversified by gene duplications. The Fur subfamily of the model fungus Aspergillus nidulans includes both major and cryptic transporters specific for uracil, 5-fluorouracil, allantoin or/and uric acid. Here we functionally analyse all four A. nidulans Fcy transporters (FcyA, FcyC, FcyD and FcyE) with previously unknown function. Our analysis shows that FcyD is moderate-affinity, low-capacity, highly specific adenine transporter, whereas FcyE contributes to 8-azaguanine uptake. Mutational analysis of FcyD, supported by homology modelling and substrate docking, shows that two variably conserved residues (Leu356 and Ser359) in transmembrane segment 8 (TMS8) are critical for transport kinetics and specificity differences among Fcy transporters, while two conserved residues(Phe167 and Ser171) in TMS3 are also important for function. Importantly, mutation S359N converts FcyD to a promiscuous nucleobase transporter capable of recognizing adenine, xanthine and several nucleobase analogues. Our results reveal the importance of specific residues in the functional evolution of NCS1 transporters.

Talaia G, Gournas C, Saliba E, Barata-Antunes C, Casal M, André B, Diallinas G, Paiva S. The α-Arrestin Bul1p Mediates Lactate Transporter Endocytosis in Response to Alkalinization and Distinct Physiological Signals. (2017) J Mol Biol. 2017 Nov 24;429(23):3678-3695. doi: 10.1016/j.jmb.2017.09.014. Epub 2017 Sep 28. PubMed PMID: 28965784.

Abstract: Eukaryotic α-arrestins connect environmental or stress signaling pathways to the endocytosis of plasma membrane transporters or receptors. The Saccharomyces cerevisiae lactate transporter Jen1p has been used as a model cargo for elucidating the mechanisms underlying endocytic turnover in response to carbon sources. Here, we discover a novel pathway of Jen1p endocytosis mediated by the α-arrestin Bul1p in response to the presence of cycloheximide or rapamycin, or prolonged growth in lactate. While cycloheximide or rapamycin modify cells pleiotropically, the major effect of prolonged growth in lactate was shown to be external pH alkalinization. Importantly, employment of specific inactive Jen1p versions showed that Bul1p-dependent endocytosis requires lactate transport, according to the signal imposed. Our results support a model where conformational changes of Jen1p, associated with substrate/H+ symport, are critical for the efficiency of Bul1p-dependent Jen1p turnover.