A SWEET surprise: Anaerobic fungal sugar transporters and chimeras enhance sugar uptake in yeast

In the yeast Saccharomyces cerevisiae, microbial fuels and chemicals production on lignocellulosic hydrolysates is constrained by poor sugar transport. For biotechnological applications, it is desirable to source transporters with novel or enhanced function from nonconventional organisms in complement to engineering known transporters. Here, we identified and functionally screened genes from three strains of early-branching anaerobic fungi (Neocallimastigomycota) that encode sugar transporters from the recently discovered Sugars Will Eventually be Exported Transporter (SWEET) superfamily in Saccharomyces cerevisiae. A novel fungal SWEET, NcSWEET1, was identified that localized to the plasma membrane and complemented growth in a hexose transporter-deficient yeast strain. Single cross-over chimeras were constructed from a leading NcSWEET1 expression-enabling domain paired with all other candidate SWEETs to broadly scan the sequence and functional space for enhanced variants. This led to the identification of a chimera, NcSW1/PfSW2:TM5-7, that enhanced the growth rate significantly on glucose, fructose, and mannose. Additional chimeras with varied cross-over junctions identified residues in TM1 that affect substrate selectivity. Furthermore, we demonstrate that NcSWEET1 and the enhanced NcSW1/PfSW2:TM5-7 variant facilitated novel co-consumption of glucose and xylose in S. cerevisiae. NcSWEET1 utilized 40.1% of both sugars, exceeding the 17.3% utilization demonstrated by the control HXT7(F79S) strain. Our results suggest that SWEETs from anaerobic fungi are beneficial tools for enhancing glucose and xylose co-utilization and offers a promising step towards biotechnological application of SWEETs in S. cerevisiae.     

Glucose transporters: physiological and pathological roles

Glucose is a primary energy source for most cells and an important substrate for many biochemical reactions. As glucose is a need of each and every cell of the body, so are the glucose transporters. Consequently, all cells express these important proteins on their surface. In recent years developments in genetics have shed new light on the types and physiology of various glucose transporters, of which there are two main types—sodium–glucose linked transporters (SGLTs) and facilitated diffusion glucose transporters (GLUT)—which can be divided into many more subclasses. Transporters differ in terms of their substrate specificity, distribution and regulatory mechanisms. Glucose transporters have also received much attention as therapeutic targets for various diseases. In this review, we attempt to present a simplified view of this complex topic which may be of interest to researchers involved in biochemical and pharmacological research.     

ABCG transporters: structure, substrate specificities and physiological roles

The ATP-binding cassette (ABC) transporter superfamily is one of the largest protein families with representatives in all kingdoms of life. Members of this superfamily are involved in a wide variety of transport processes with substrates ranging from small ions to relatively large polypeptides and polysaccharides. The G subfamily of ABC transporters consists of half-transporters, which oligomerise to form the functional transporter. While ABCG1, ABCG4 and ABCG5/8 are involved in the ATP-dependent translocation of steroids and, possibly, other lipids, ABCG2 (also termed the breast cancer resistance protein) has been identified as a multidrug transporter that confers resistance on tumor cells. Evidence will be summarized suggesting that ABCG2 can also mediate the binding/transport of non-drug substrates, including free and conjugated steroids. The characterization of the substrate specificities of ABCG proteins at a molecular level might provide further clues about their potential physiological role(s), and create new opportunities for the modulation of their activities in relation to human disease.     

Structure, function and regulation of ammonium transporters in plants

Ammonium is an important source of nitrogen for plants. It is taken up by plant cells via ammonium transporters in the plasma membrane and distributed to intracellular compartments such as chloroplasts, mitochondria and vacuoles probably via different transporters in each case. Ammonium is generally not used for long-distance transport of nitrogen within the plant. Instead, most of the ammonium transported into plant cells is assimilated locally via glutamine synthetases in the cytoplasm and plastids. Ammonium is also produced by plant cells during normal metabolism, and ammonium transporters enable it to be moved from intracellular sites of production to sites of consumption. Ammonium can be generated de novo from molecular nitrogen (N(2)) by nitrogen-fixing bacteria in some plant cells, such as rhizobia in legume root nodule cells, and at least one ammonium transporter is implicated in the transfer of ammonium from the bacteria to the plant cytoplasm. Plant physiologists have described many of these ammonium transport processes over the last few decades. However, the genes and proteins that underlie these processes have been isolated and studied only recently. In this review, we consider in detail the molecular structure, function and regulation of plant ammonium transporters. We also attempt to reconcile recent discoveries at the molecular level with our knowledge of ammonium transport at the whole plant level.     

Structure and function of facilitative sugar transporters.

Sugar transporters from one group of the major facilitator superfamily of membrane transporters. A conserved common central pore structure lies at the heart of these transporters and diverse functionality is brought about by alterations to this pore or regions associated with it. Recent mutagenesis studies of sugar transporters within the framework of tenable models for the distantly related lactose permease argue that this model is a good paradigm for other members of the major facilitator superfamily.     

Bacterial ATP-driven transporters of transition metals: physiological roles, mechanisms of action, and roles in bacterial virulence

Maintaining adequate intracellular levels of transition metals is fundamental to the survival of all organisms. While all transition metals are toxic at elevated intracellular concentrations, metals such as iron, zinc, copper, and manganese are essential to many cellular functions. In prokaryotes, the concerted action of a battery of membrane-embedded transport proteins controls a delicate balance between sufficient acquisition and overload. Representatives from all major families of transporters participate in this task, including ion-gradient driven systems and ATP-utilizing pumps. P-type ATPases and ABC transporters both utilize the free energy of ATP hydrolysis to drive transport. Each of these very different families of transport proteins has a distinct role in maintaining transition metal homeostasis: P-type ATPases prevent intracellular overloading of both essential and toxic metals through efflux while ABC transporters import solely the essential ones. In the present review we discuss how each system is adapted to perform its specific task from mechanistic and structural perspectives. Despite the mechanistic and structural differences between P-type ATPases and ABC transporters, there is one important commonality: in many clinically relevant bacterial pathogens, transporters of transition metals are essential for virulence. Here we present several such examples and discuss how these may be exploited for future antibacterial drug development.     

Genome-wide analysis indicates diverse physiological roles of the turnip (Brassica rapa var. rapa) oligopeptide transporters gene family

Oligopeptide transporters (OPTs) encode integral membrane-localized proteins and have a broad range of substrate transport capabilities.Here,28 BrrOPT genes were identified in the turnip.Phylogenetic analyses revealed two well-supported clades in the OPT family,containing 15 BrrOPTs and 13 BrrYSLs. The exon/intron structure of OPT clade are conserved but the yellow stripe-like (YSL) clade was different. The exon/intron of the YSL clade possesses structural differences,whereas the YSL class motifs structure are conserved.The OPT genes are distributed unevenly among the chromosomes of the turnip genome. Phylogenetic and chromosomal distribution analyses revealed that the expansion of the OPT gene family is mainly attributable to segmental duplication.For the expression profiles at different developmental stages,a comprehensive analysis provided insights into the possible functional divergence among members of the paralog OPT gene family.Different expression levels under a variety of ion deficiencies also indicated that the OPT family underwent functional divergence during long-term evolution. Furthermore,BrrOPT8.1,BrrYSL1.2,BrrYSL1.3,BrrYSL6 and BrrYSL9 responded to Fe (Ⅱ) treatments and BrrYSL7 responded to calcium treatments,BrrYSL6 responded to multiple treatments in root,suggesting that turnip OPTs may be involved in mediating cross-talk among different ion deficiencies.Our data provide important information for further functional dissection of BrrOPTs,especially in transporting metal ions and nutrient deficiency stress adaptation.     

Endocytosis in plants: Peculiarities and roles in the regulated trafficking of plant metal transporters

The removal of transmembrane proteins from the plasma membrane via endocytosis has emerged as powerful strategy in the regulation of receptor signalling and molecule transport. In the last decade, IRON‐REGULATED TRANSPORTER1 (IRT1) has been established as one of the key plant model proteins for studying endomembrane trafficking. The use of IRT1 and additional other metal transporters has uncovered novel factors involved in plant endocytosis and facilitated a better understanding of the role of endocytosis in the fine balancing of plant metal homoeostasis. In this review, we outline the specifics of plant endocytosis compared to what is known in yeast and mammals, and based on several examples, we demonstrate how studying metal transport has contributed to extending our knowledge of endocytic trafficking by shedding light on novel regulatory mechanisms and factors.     

Stochastic proton pumping ATPases: from single molecules to diverse physiological roles

We discuss the most recent reports on two proton pumps, F-ATPase (ATP synthase) and V-ATPase (endomembrane proton pump). They are formed from similar extrinsic (F1 or V1) and intrinsic (Fo or Vo) membrane sectors, and couple chemistry and proton transport through subunit rotation for apparently different physiological roles. Emphasis is placed on the stochastic rotational catalysis of F-ATPase and isoforms of V-ATPase.     

Structural foundations for explaining the physiological roles of murzymes embedded in diverse phospholipid membranes

The advent of improved structural biology protocols and bioinformatics methodologies have provided paradigm-shifting insights on metabolic or physiological processes catalyzed by homo?/hetero- proteins (super)complexes embedded in phospholipid membranes of cells/organelles. In this panoramic review, we succinctly elucidate the structural features of select redox proteins from four systems: hepatocyte/adrenal cortex endoplasmic reticulum (microsomes), inner mitochondrial membrane (cristae), thylakoid membrane (grana), and in the flattened disks of rod/cone cells (in retina). Besides catalyzing fast/crucial (photo)chemical reactions, these proteins utilize the redox-active diatomic gaseous molecule of oxygen, the elixir of aerobic life. Quite contrary to extant perceptions that invoke primarily deterministic affinity-binding or conformation-change based “proton-pump”/“serial electron-relay” type roles, we advocate murzyme functions for the membrane-embedded proteins in these systems. Murzymes are proteins that generate/stabilize/utilize diffusible reactive (oxygen) species (DRS/DROS) based activities. Herein, we present a brief compendium of the recently revealed wealth of structural information and mechanistic concepts on how the membrane proteins use DRS/DROS to aid ‘effective charge separation’ and facilitate trans-membrane dynamics of diverse species in milieu, thereby enabling the cells to function as ‘simple chemical engines’.     

GLUT,SGLT, and SWEET: Structural and mechanistic investigations of the glucose transporters

Glucose is the primary fuel to life on earth. Cellular uptake of glucose is a fundamental process for metabolism, growth, and homeostasis. Three families of secondary glucose transporters have been identified in human, including the major facilitator superfamily glucose facilitators GLUTs, the sodium‐driven glucose symporters SGLTs, and the recently identified SWEETs. Structures of representative members or their prokaryotic homologs of all three families were obtained. This review focuses on the recent advances in the structural elucidation of the glucose transporters and the mechanistic insights derived from these structures, including the molecular basis for substrate recognition, alternating access, and stoichiometric coupling of co‐transport.     

The diverse roles of ubiquitin and the 26S proteasome in the life of plants

A tightly regulated and highly specific system for the degradation of individual proteins is essential for the survival of all organisms. In eukaryotes, this is achieved by the tagging of proteins with ubiquitin and their subsequent recognition and degradation by the 26S proteasome. In plants, genetic analysis has identified many genes that regulate developmental pathways. Subsequent analysis of these genes has implicated ubiquitin and the 26S proteasome in the control of diverse developmental processes, and indicates that proteolysis is a crucial regulatory step throughout the life cycle of plants.     

Structure, function, and evolution of the PsbP protein family in higher plants

The PsbP is a thylakoid lumenal subunit of photosystem II (PSII), which has developed specifically in higher plants and green algae. In higher plants, the molecular function of PsbP has been intensively investigated by release-reconstitution experiments in vitro. Recently, solution of a high-resolution structure of PsbP has enabled investigation of structure-function relationships, and efficient gene-silencing techniques have demonstrated the crucial role of PsbP in PSII activity in vivo. Furthermore, genomic and proteomic studies have shown that PsbP belongs to the divergent PsbP protein family, which consists of about 10 members in model plants such as Arabidopsis and rice. Characterization of the molecular function of PsbP homologs using Arabidopsis mutants suggests that each plays a distinct and important function in maintaining photosynthetic electron transfer. In this review, recent findings regarding the molecular functions of PsbP and other PsbP homologs in higher plants are summarized, and the molecular evolution of these proteins is discussed.