Strategies for Efficient Expression of Heterologous Monosaccharide Transporters in Saccharomyces cerevisiae

In previous work, we developed a Saccharomyces cerevisiae strain (DLG-K1) lacking the main monosaccharide transporters (hxt-null) and displaying high xylose reductase, xylitol dehydrogenase and xylulokinase activities. This strain proved to be a useful chassis strain to study new glucose/xylose transporters, as SsXUT1 from Scheffersomyces stipitis. Proteins with high amino acid sequence similarity (78–80%) to SsXUT1 were identified from Spathaspora passalidarum and Spathaspora arborariae genomes. The characterization of these putative transporter genes (SpXUT1 and SaXUT1, respectively) was performed in the same chassis strain. Surprisingly, the cloned genes could not restore the ability to grow in several monosaccharides tested (including glucose and xylose), but after being grown in maltose, the uptake of 14C-glucose and 14C-xylose was detected. While SsXUT1 lacks lysine residues with high ubiquitinylation potential in its N-terminal domain and displays only one in its C-terminal domain, both SpXUT1 and SaXUT1 transporters have several such residues in their C-terminal domains. A truncated version of SpXUT1 gene, deprived of the respective 3′-end, was cloned in DLG-K1 and allowed growth and fermentation in glucose or xylose. In another approach, two arrestins known to be involved in the ubiquitinylation and endocytosis of sugar transporters (ROD1 and ROG3) were knocked out, but only the rog3 mutant allowed a significant improvement of growth and fermentation in glucose when either of the XUT permeases were expressed. Therefore, for the efficient heterologous expression of monosaccharide (e.g., glucose/xylose) transporters in S. cerevisiae, we propose either the removal of lysines involved in ubiquitinylation and endocytosis or the use of chassis strains hampered in the specific mechanism of membrane protein turnover.

Transcription Factor CitERF16 Is Involved in Citrus Fruit Sucrose Accumulation by Activating CitSWEET11d

Sugars are the primary products of photosynthesis and play an important role in plant growth and development. They contribute to sweetness and flavor of fleshy fruits and are pivotal to fruit quality, and their translocation and allocation are mainly dependent on sugar transporters. Genome-wide characterization of Satsuma mandarin identified eighteen SWEET family members that encode transporters which facilitate diffusion of sugar across cell membranes. Analysis of the expression profiles in tissues of mandarin fruit at different developmental stages showed that CitSWEET11d transcripts were significantly correlated with sucrose accumulation. Further studies indicated that overexpression of CitSWEET11d in citrus callus and tomato fruit showed a higher sucrose level compared to wild-type, suggesting that CitSWEET11d could enhance sucrose accumulation. In addition, we identified an ERF transcription factor CitERF16 by yeast one-hybrid screening assay which could directly bind to the DRE cis-element on the promoter of CitSWEET11d. Overexpression of CitERF16 in citrus callus significantly induced CitSWEET11d expression and elevated sucrose content, suggesting that CitERF16 acts as a positive regulator to promote sucrose accumulation via trans-activation of CitSWEET11d expression.

When SWEETs Turn Tweens: Updates and Perspectives

Sugar translocation between cells and between subcellular compartments in plants requires either plasmodesmata or a diverse array of sugar transporters. Interactions between plants and associated microorganisms also depend on sugar transporters. The sugars will eventually be exported transporter (SWEET) family is made up of conserved and essential transporters involved in many critical biological processes. The functional significance and small size of these proteins have motivated crystallographers to successfully capture several structures of SWEETs and their bacterial homologs in different conformations. These studies together with molecular dynamics simulations have provided unprecedented insights into sugar transport mechanisms in general and into substrate recognition of glucose and sucrose in particular. This review summarizes our current understanding of the SWEET family, from the atomic to the whole-plant level. We cover methods used for their characterization, theories about their evolutionary origins, biochemical properties, physiological functions, and regulation. We also include perspectives on the future work needed to translate basic research into higher crop yields. Expected final online publication date for the Annual Review of Plant Biology, Volume 73 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Genetic evidence that uptake of the fluorescent analog 2NBDG occurs independently of known glucose transporters

The fluorescent derivative of glucose, 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]-D-glucose (2NBDG), is a widely used surrogate reagent to visualize glucose uptake in live cells at single cell resolution. Using a model of CRISPR-Cas9 gene editing in 5TGM1 myeloma cells, we demonstrate that ablation of the glucose transporter gene Slc2a1 abrogates radioactive glucose uptake but has no effect on the magnitude or kinetics of 2NBDG import. Extracellular 2NBDG, but not NBD-fructose was transported by plasma cells into the cytoplasm suggesting specific activity that is unlinked to glucose import and that of chemically similar compounds. RNA-Seq analysis of primary plasma cells and the 5TGM1 myeloma cell line revealed expression of other candidate glucose transporters. Yet, deletion of these transporters individually or in combination with one another also had no impact on 2NBDG uptake. Ablation of the genes in the Slc29 and Slc35 families of nucleoside and nucleoside sugar transporters as well as the ATP-binding cassette (ABC) transporter family also failed to impact 2NBDG import. Thus, cellular uptake of 2NBDG is promoted by an unknown mechanism and is not a faithful indicator of glucose transport.

Sugar transporters of the SWEET family and their role in arbuscular mycorrhiza

Plant sugar transporters play an essential role in the organism’s productivity by carrying out carbohydrate transportation from source cells in the leaves to sink cells in the cortex. In addition, they aid in the regulation of a substantial part of the exchange of nutrients with microorganisms in the rhizosphere (bacteria and fungi), an activity essential to the formation of symbiotic relationships. This review pays special attention to carbohydrate nutrition during the development of arbuscular mycorrhiza (AM), a symbiosis of plants with fungi from the Glomeromycotina subdivision. This relationship results in the host plant receiving micronutrients from the mycosymbiont, mainly phosphorus, and the fungus receiving carbon assimilation products in return. While the efficient nutrient transport pathways in AM symbiosis are yet to be discovered, SWEET sugar transporters are one of the three key families of plant carbohydrate transporters. Specific AM symbiosis transporters can be identified among the SWEET proteins. The survey provides data on the study history, structure and localization, phylogeny and functions of the SWEET proteins. A high variability of both the SWEET proteins themselves and their functions is noted along with the fact that the same proteins may perform different functions in different plants. A special role is given to the SWEET transporters in AM development. SWEET transporters can also play a key role in abiotic stress tolerance, thus allowing plants to adapt to adverse environmental conditions. The development of knowledge about symbiotic systems will contribute to the creation of microbial preparations for use in agriculture in the Russian Federation.

New Strategies for Efficient Expression of Heterologous Sugar Transporters in Saccharomyces cerevisiae

: In our previous work we had developed an hxt-null Saccharomyces cerevisiae strain displaying high xylose reductase, xylitol dehydrogenase and xylulokinase activities that proved to be useful as a chassis strain to study new xylose transporters, as SsXUT1 from Scheffersomyces stipitis. Spathaspora passalidarum and Spathaspora arborariae have in their genomes genes with high sequence similarity (78-80%) to SsXUT1. To characterize these putative transporter genes (SpXUT1 and SaXUT1, respectively) they were expressed in the same chassis strain as SsXUT1. Surprisingly, the cloned genes could not restore the ability to grow in several monosaccharides tested, although the strains expressing the SsXUT1 and SpXUT1 permeases, after growth on maltose, showed the presence of 14C-glucose and 14C-xylose transport activity. An important feature of these permeases is that SsXUT1 lacks lysine residues in its N-terminal domain with high-confidence ubiquitinylation potential, and has only one at the C-terminal domain, while the SpXUT1 transporter had several of such residues at its C-terminal domain. When the SpXUT1 gene was cloned in a truncated version lacking such lysine residues, the permease allowed grow on glucose or xylose, and even promoted xylose fermentation by the hxt-null strain. In another approach, we deleted two arrestins known to be involved in sugar transporter ubiquitinylation and endocytosis (ROD1 and ROG3), but only the rog3Δ strain allowed modest growth on these sugars. Taken together, these results suggest that to allow efficient sugar transporter expression in S. cerevisiae the lysines involved in transporter endocytosis should be removed from the sequence of the permease.

Getting Sugar Coating Right! The Role of the Golgi Trafficking Machinery in Glycosylation

The Golgi is the central organelle of the secretory pathway and it houses the majority of the glycosylation machinery, which includes glycosylation enzymes and sugar transporters. Correct compartmentalization of the glycosylation machinery is achieved by retrograde vesicular trafficking as the secretory cargo moves forward by cisternal maturation. The vesicular trafficking machinery which includes vesicular coats, small GTPases, tethers and SNAREs, play a major role in coordinating the Golgi trafficking thereby achieving Golgi homeostasis. Glycosylation is a template-independent process, so its fidelity heavily relies on appropriate localization of the glycosylation machinery and Golgi homeostasis. Mutations in the glycosylation enzymes, sugar transporters, Golgi ion channels and several vesicle tethering factors cause congenital disorders of glycosylation (CDG) which encompass a group of multisystem disorders with varying severities. Here, we focus on the Golgi vesicle tethering and fusion machinery, namely, multisubunit tethering complexes and SNAREs and their role in Golgi trafficking and glycosylation. This review is a comprehensive summary of all the identified CDG causing mutations of the Golgi trafficking machinery in humans.

Insights into fucose metabolism: SLC35C1-independent fucosylation discriminates against mannose-derived GDP-fucose

Mutations in the SLC35C1 gene, encoding the Golgi GDP-fucose transporter, cause leukocyte adhesion deficiency II (LADII). Fucosylation improvement in LADII patients treated with fucose suggests the existence of an SLC35C1-independent route of GDP-fucose transport, which still remains a mystery. Here, we developed and characterized a human cell-based model deficient in the SLC35C1 activity. The knockout cells displayed low but detectable levels of fucosylation. Strikingly, the fucosylation defect was almost completely reversed upon treatment with millimolar concentrations of fucose. Even if fucose was supplemented at nanomolar concentrations, it was still incorporated into glycans by the knockout cells. We also found that the SLC35C1-independent transport preferred the salvage pathway over the de novo pathway as a source of GDP-fucose. Our results imply that the Golgi systems of GDP-fucose transport discriminate between the substrate pools obtained from different metabolic pathways, which suggests a functional connection between nucleotide sugar transporters and nucleotide sugar synthetases.