Remembering Otto Kandler (1920-2017) and his contributions.

After a brief prologue on Otto Kandler's life, we describe briefly his pioneering work on photosynthesis (photophosphorylation and the carbon cycle) and his key participation in the discovery of the concept of three forms of life (Archaea, Prokarya, and Eukarya). With Otto Kandler's passing, both the international photosynthesis and microbiology communities have lost an internationally unique, eminent, and respected researcher and teacher who exhibited a rare vibrancy and style.

Transmembrane voltage: Potential to induce lateral microdomains.

Lateral segregation of plasma membrane lipids is a generally accepted phenomenon. Lateral lipid microdomains of specific composition, structure and biological functions are established as a result of simultaneous action of several competing mechanisms which contribute to membrane organization. Various lines of evidence support the conclusion that among those mechanisms, the membrane potential plays significant and to some extent unique role. Above all, clear differences in the microdomain structure as revealed by fluorescence microscopy could be recognized between polarized and depolarized membranes. In addition, recent fluorescence spectroscopy experiments reported depolarization-induced changes in a membrane lipid order. In the context of earlier findings showing that plasma membranes of depolarized cells are less susceptible to detergents and the cells less sensitive to antibiotics or antimycotics treatment we discuss a model, in which membrane potential-driven re-organization of the microdomain structure contributes to maintaining membrane integrity during response to stress, pathogen attack and other challenges involving partial depolarization of the plasma membrane. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Transport of sugars.

Soluble sugars serve five main purposes in multicellular organisms: as sources of carbon skeletons, osmolytes, signals, and transient energy storage and as transport molecules. Most sugars are derived from photosynthetic organisms, particularly plants. In multicellular organisms, some cells specialize in providing sugars to other cells (e.g., intestinal and liver cells in animals, photosynthetic cells in plants), whereas others depend completely on an external supply (e.g., brain cells, roots and seeds). This cellular exchange of sugars requires transport proteins to mediate uptake or release from cells or subcellular compartments. Thus, not surprisingly, sugar transport is critical for plants, animals, and humans. At present, three classes of eukaryotic sugar transporters have been characterized, namely the glucose transporters (GLUTs), sodium-glucose symporters (SGLTs), and SWEETs. This review presents the history and state of the art of sugar transporter research, covering genetics, biochemistry, and physiology-from their identification and characterization to their structure, function, and physiology. In humans, understanding sugar transport has therapeutic importance (e.g., addressing diabetes or limiting access of cancer cells to sugars), and in plants, these transporters are critical for crop yield and pathogen susceptibility.

Membrane microdomains, rafts, and detergent-resistant membranes in plants and fungi.

The existence of specialized microdomains in plasma membranes, postulated for almost 25 years, has been popularized by the concept of lipid or membrane rafts. The idea that detergent-resistant membranes are equivalent to lipid rafts, which was generally abandoned after a decade of vigorous data accumulation, contributed to intense discussions about the validity of the raft concept. The existence of membrane microdomains, meanwhile, has been verified by unequivocal independent evidence. This review summarizes the current state of research in plants and fungi with respect to common aspects of both kingdoms. In these organisms, principally immobile microdomains large enough for microscopic detection have been visualized. These microdomains are found in the context of cell-cell interactions (plant symbionts and pathogens), membrane transport, stress, and polarized growth, and the data corroborate at least three mechanisms of formation. As documented in this review, modern methods of visualization of lateral membrane compartments are also able to uncover the functional relevance of membrane microdomains.

Distribution of cortical endoplasmic reticulum determines positioning of endocytic events in yeast plasma membrane.

In many eukaryotes, a significant part of the plasma membrane is closely associated with the dynamic meshwork of cortical endoplasmic reticulum (cortical ER). We mapped temporal variations in the local coverage of the yeast plasma membrane with cortical ER pattern and identified micron-sized plasma membrane domains clearly different in cortical ER persistence. We show that clathrin-mediated endocytosis is initiated outside the cortical ER-covered plasma membrane zones. These cortical ER-covered zones are highly dynamic but do not overlap with the immobile and also endocytosis-inactive membrane compartment of Can1 (MCC) and the subjacent eisosomes. The eisosomal component Pil1 is shown to regulate the distribution of cortical ER and thus the accessibility of the plasma membrane for endocytosis.

The lateral compartmentation of the yeast plasma membrane.

The plasma membrane of Saccharomyces cerevisiae contains large microdomains enriched in ergosterol, which house at least nine integral proteins, including proton symporters. The domains adopt a characteristic structure of furrow-like invaginations typically seen in freeze-fracture pictures of fungal cells. Being stable for the time comparable with the cell cycle duration, they might be considered as fixed islands (rafts) in an otherwise fluid yeast plasma membrane. Rapidly moving endocytic marker proteins avoid the microdomains; the domain-accumulated proton symporters consequently show a reduced rate of substrate-induced endocytosis and turnover.

C terminus of Nce102 determines the structure and function of microdomains in the Saccharomyces cerevisiae plasma membrane.

The plasma membrane of the yeast Saccharomyces cerevisiae contains stably distributed lateral domains of specific composition and structure, termed MCC (membrane compartment of arginine permease Can1). Accumulation of Can1 and other specific proton symporters within MCC is known to regulate the turnover of these transporters and is controlled by the presence of another MCC protein, Nce102. We show that in an NCE102 deletion strain the function of Nce102 in directing the specific permeases into MCC can be complemented by overexpression of the NCE102 close homolog FHN1 (the previously uncharacterized YGR131W) as well as by distant Schizosaccharomyces pombe homolog fhn1 (SPBC1685.13). We conclude that this mechanism of plasma membrane organization is conserved through the phylum Ascomycota. We used a hemagglutinin (HA)/Suc2/His4C reporter to determine the membrane topology of Nce102. In contrast to predictions, its N and C termini are oriented toward the cytosol. Deletion of the C terminus or even of its last 6 amino acids does not disturb protein trafficking, but it seriously affects the formation of MCC. We show that the C-terminal part of the Nce102 protein is necessary for localization of both Nce102 itself and Can1 to MCC and also for the formation of furrow-like membrane invaginations, the characteristic ultrastructural feature of MCC domains.

Furrow-like invaginations of the yeast plasma membrane correspond to membrane compartment of Can1.

Plasma membrane of the yeast Saccharomyces cerevisiae contains stable lateral domains. We have investigated the ultrastructure of one type of domain, the membrane compartment of Can1 (MCC). In two yeast strains (nce102Delta and pil1Delta) that are defective in segregation of MCC-specific proteins, we found the plasma membrane to be devoid of the characteristic furrow-like invaginations. These are highly conserved plasma membrane structures reported in early freeze-fracture studies. Comparison of the results obtained by three different approaches - electron microscopy of freeze-etched cells, confocal microscopy of intact cells and computer simulation - shows that the number of invaginations corresponds to the number of MCC patches in the membrane of wild-type cells. In addition, neither MCC patches nor the furrow-like invaginations colocalized with the cortical ER. In mutants exhibiting elongated MCC patches, there are elongated invaginations of the appropriate size and frequency. Using various approaches of immunoelectron microscopy, the MCC protein Sur7, as well as the eisosome marker Pil1, have been detected at these invaginations. Thus, we identify the MCC patch, which is a lateral membrane domain of specific composition and function, with a specific structure in the yeast plasma membrane - the furrow-like invagination.

Plasma membrane microdomains regulate turnover of transport proteins in yeast.

In this study, we investigate whether the stable segregation of proteins and lipids within the yeast plasma membrane serves a particular biological function. We show that 21 proteins cluster within or associate with the ergosterol-rich membrane compartment of Can1 (MCC). However, proteins of the endocytic machinery are excluded from MCC. In a screen, we identified 28 genes affecting MCC appearance and found that genes involved in lipid biosynthesis and vesicle transport are significantly overrepresented. Deletion of Pil1, a component of eisosomes, or of Nce102, an integral membrane protein of MCC, results in the dissipation of all MCC markers. These deletion mutants also show accelerated endocytosis of MCC-resident permeases Can1 and Fur4. Our data suggest that release from MCC makes these proteins accessible to the endocytic machinery. Addition of arginine to wild-type cells leads to a similar redistribution and increased turnover of Can1. Thus, MCC represents a protective area within the plasma membrane to control turnover of transport proteins.

Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast.

The plasma membrane potential is mainly considered as the driving force for ion and nutrient translocation. Using the yeast Saccharomyces cerevisiae as a model organism, we have discovered a novel role of the membrane potential in the organization of the plasma membrane. Within the yeast plasma membrane, two non-overlapping sub-compartments can be visualized. The first one, represented by a network-like structure, is occupied by the proton ATPase, Pma1, and the second one, forming 300-nm patches, houses a number of proton symporters (Can1, Fur4, Tat2 and HUP1) and Sur7, a component of the recently described eisosomes. Evidence is presented that sterols, the main lipid constituent of the plasma membrane, also accumulate within the patchy compartment. It is documented that this compartmentation is highly dependent on the energization of the membrane. Plasma membrane depolarization causes reversible dispersion of the H(+)-symporters, not however of the Sur7 protein. Mitochondrial mutants, affected in plasma membrane energization, show a significantly lower degree of membrane protein segregation. In accordance with these observations, depolarized membranes also considerably change their physical properties (detergent sensitivity).

Protein glycosylation, conserved from yeast to man: a model organism helps elucidate congenital human diseases.

Proteins can be modified by a large variety of covalently linked saccharides. The present review concentrates on two types, protein N-glycosylation and protein O-mannosylation, which, with only a few exceptions, are evolutionary conserved from yeast to man. They are also distinguished by some special features: The corresponding glycosylation processes start in the endoplasmatic reticulum, are continued in the Golgi apparatus, and require dolichol-activated precursors for the initial biosynthetic steps. With respect to the molecular biology of both types of protein glycosylation, the pathways and the genetic background of the reactions have most successfully been studied with the genetically easy-to-handle baker's yeast, Saccharomyces cerevisae. Many of the severe developmental disturbances in children are related to protein glycosylation, for example, the CDG syndrome (congenital disorders of glycosylation) as well as congenital muscular dystrophies with neuronal-cell-migration defects have been elucidated with the help of yeast.

Lipid raft-based membrane compartmentation of a plant transport protein expressed in Saccharomyces cerevisiae.

The hexose-proton symporter HUP1 shows a spotty distribution in the plasma membrane of the green alga Chlorella kessleri. Chlorella cannot be transformed so far. To study the membrane localization of the HUP1 protein in detail, the symporter was fused to green fluorescent protein (GFP) and heterologously expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In these organisms, the HUP1 protein has previously been shown to be fully active. The GFP fusion protein was exclusively targeted to the plasma membranes of both types of fungal cells. In S. cerevisiae, it was distributed nonhomogenously and concentrated in spots resembling the patchy appearance observed previously for endogenous H(+) symporters. It is documented that the Chlorella protein colocalizes with yeast proteins that are concentrated in 300-nm raft-based membrane compartments. On the other hand, it is completely excluded from the raft compartment housing the yeast H(+)/ATPase. As judged by their solubilities in Triton X-100, the HUP1 protein extracted from Chlorella and the GFP fusion protein extracted from S. cerevisiae are detergent-resistant raft proteins. S. cerevisiae mutants lacking the typical raft lipids ergosterol and sphingolipids showed a homogenous distribution of HUP1-GFP within the plasma membrane. In an ergosterol synthesis (erg6) mutant, the rate of glucose uptake was reduced to less than one-third that of corresponding wild-type cells. In S. pombe, the sterol-rich plasma membrane domains can be stained in vivo with filipin. Chlorella HUP1-GFP accumulated exactly in these domains. Altogether, it is demonstrated here that a plant membrane protein has the property of being concentrated in specific raft-based membrane compartments and that the information for its raft association is retained between even distantly related organisms.

Pir proteins of Saccharomyces cerevisiae are attached to beta-1,3-glucan by a new protein-carbohydrate linkage.

A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. The exchange of each of three glutamines (Gln69, Gln74, Gln76) as well as one aspartic acid (Asp72) within the repeating unit leads to a loss of the protein from the cell wall. Amino acid sequencing revealed that only Gln74 is modified. Release of the protein with mild alkali, changed Gln74 to to glutamic acid, suggesting that Gln74 is involved in the linkage. Analysis by mass spectrometry showed that 5 hexoses are attached to Gln/Glu74. Sugar analysis revealed glucose as the only constituent. It is suggested that Pir proteins form novel, alkali labile ester linkages between the gamma-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of beta-1,3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination.

Differential effect of phosphatidylethanolamine depletion on raft proteins: further evidence for diversity of rafts in Saccharomyces cerevisiae.

A considerable amount of evidence supports the idea that lipid rafts are involved in many cellular processes, including protein sorting and trafficking. We show that, in this process, also a non-raft lipid, phosphatidylethanolamine (PE), has an indispensable function. The depletion of this phospholipid results in an accumulation of a typical raft-resident, the arginine transporter Can1p, in the membranes of Golgi, while the trafficking of another plasma membrane transporter, Pma1p, is interrupted at the level of the ER. Both these transporters associate with a Triton (TX-100) resistant membrane fraction before their intracellular transport is arrested in the respective organelles. The Can1p undelivered to the plasma membrane is fully active when reconstituted to a PE-containing vesicle system in vitro. We further demonstrate that, in addition to the TX-100 resistance at 4 degrees C, Can1p and Pma1pa exhibit different accessibility to nonyl glucoside (NG), which points to distinct intimate lipid surroundings of these two proteins. Also, at 20 degrees C, these two proteins are extracted by TX-100 differentially. The features above suggest that Pma1p and Can1p are associated with different compartments. This is independently supported by the observations made by confocal microscopy. In addition we show that PE is involved in the stability of Can1p-raft association.

Distribution of Can1p into stable domains reflects lateral protein segregation within the plasma membrane of living S. cerevisiae cells.

Recently, lipid-raft-based subdomains within the plasma membrane of living Saccharomyces cerevisiae cells were visualized using green fluorescent protein fusions, and non-overlapping subdomains containing either Pma1p or Can1p were distinguished. In this study, the long-term stability of the subdomains was investigated. Experiments with latrunculin A and nocodazole ruled out the involvement of cytoskeletal components in the stabilization of the subdomains. Also a putative role of the cell wall was excluded, because protoplasting of the cells changed neither the pattern nor the stability of the subdomains. By contrast, the expected inner dynamics of the membrane subdomains was documented by FRAP experiments. Finally, two other proteins were localized within the frame of the Can1p/Pma1p plasma-membrane partition. We show that Fur4p (another H+ symporter) and Sur7p (a protein of unknown function) occupy the Can1p subdomain.

Scw10p, a cell-wall glucanase/transglucosidase important for cell-wall stability in Saccharomyces cerevisiae.

Glycosyl hydrolases and transferases are crucial for the formation of a rigid but at the same time plastic cell wall in yeasts and fungi. The Saccharomyces cerevisiae glucan hydrolase family 17 (GH17) contains the soluble cell-wall proteins Scw4p, Scw10p, Scw11p and Bgl2p. For Bgl2p, endoglucanase/glucanosyltransferase activity has been demonstrated, and Scw11p has been shown to be involved in cell separation. Here, Scw4p and Scw10p, which show 63 % amino acid identity, were characterized. scw4 and scw10 single mutants were sensitive towards cell-wall destabilizing agents, suggesting a role in cell-wall assembly or maintenance. Simultaneous deletion of SCW4 and SCW10 showed a synergistic effect, and activated the cell-wall compensatory mechanism in a PKC1-dependent manner. Both the amount of cell-wall chitin and the amount of mannoproteins attached to chitin were increased in mutant scw4scw10. Deletion of CHS3 proved the critical role of chitin in scw4scw10. However, the mannoprotein Sed1p and the glucan synthase Fks2p were also crucial for cell-wall stability in mutant scw4scw10. The exchange of two conserved glutamate residues localized in the putative catalytic domain of GH17 family members strongly suggests that Scw10p acts as a 1,3-beta-glucanase or as a 1,3-beta-glucanosyltransferase. In addition, the synthetic interactions between Bgl2p and Scw10p which support a functional cooperation in cell-wall assembly were analysed. The data suggest that Scw4p and Scw10p act as glucanases or transglucosidases in concert with other cell-wall proteins to assure cell-wall integrity.

Targeted disruption of the Walker-Warburg syndrome gene Pomt1 in mouse results in embryonic lethality.

O-mannosylation is an important protein modification in eukaryotes that is initiated by an evolutionarily conserved family of protein O-mannosyltransferases. The first mammalian protein O-mannosyltransferase gene described was the human POMT1. Mutations in the hPOMT1 gene are responsible for Walker-Warburg syndrome (WWS), a severe recessive congenital muscular dystrophy associated with defects in neuronal migration that produce complex brain and eye abnormalities. During embryogenesis, the murine Pomt1 gene is prominently expressed in the neural tube, the developing eye, and the mesenchyme. These sites of expression correlate with those in which the main tissue alterations are observed in WWS patients. We have inactivated a Pomt1 allele by gene targeting in embryonic stem cells and produced chimeras transmitting the defect allele to offspring. Although heterozygous mice were viable and fertile, the total absence of Pomt1(-/-) pups in the progeny of heterozygous intercrosses indicated that this genotype is embryonic lethal. An analysis of the mutant phenotype revealed that homozygous Pomt1(-/-) mice suffer developmental arrest around embryonic day (E) 7.5 and die between E7.5 and E9.5. The Pomt1(-/-) embryos present defects in the formation of Reichert's membrane, the first basement membrane to form in the embryo. The failure of this membrane to form appears to be the result of abnormal glycosylation and maturation of dystroglycan that may impair recruitment of laminin, a structural component required for the formation of Reichert's membrane in rodents. The targeted disruption of mPomt1 represents an example of an engineered deletion of a known glycosyltransferase involved in O-mannosyl glycan synthesis.