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In 75 semesters, from mannan and dolichol to Pir proteins and membrane compartmentation: personal recollections

2007; Wiley; Volume: 24; Issue: 4 Linguagem: Inglês

10.1002/yea.1474

1097-0061

Widmar Tanner,

Lipid metabolism and biosynthesis

Introduction The past The present The future References When the organizers of the 3rd International Conference on Molecular Mechanisms of Fungal Cell Wall Biogenesis (Heidelberg, 2006) asked me half a year after my official retirement to give a special lecture, I assumed that I should speak about the past. Therefore, I will start these personal recollections with a somewhat longer section about my scientific past, which appears to be closer to one's heart, the older one gets. Then I will report about my present and potential future work. When I tried to recall why and how I started to work with baker's yeast, I realized how completely different science is nowadays, with genome sequences and mutant collections available and computers to hand. Thus, I guess it might be interesting for young researchers to hear how their scientific grandfathers approached problems and obtained results, about which they were at least as excited as students are nowadays. Of course, I experienced Saccharomyces cerevisiae first in biology classes. But I was considerably more impressed when I came across yeast cells in Feodor Lynen's ‘Biochemisches Grosspraktikum’, where one of my tasks was to isolate and purify NAD+ (at that time called DPN+) from one pound of baker's yeast. In the course of this ‘Grosspraktikum’ and its exam-like discussions, which we had with our teaching assistant Guido Hartmann every Saturday morning, we also learnt of Lynen's research. We came to know how he and his co-workers elucidated the complete pathway of fatty acid β-oxidation in baker's yeast, as well as the biosynthesis of isopentenyl-pyrophosphate and its role in cholesterol biosynthesis, which earned Lynen the Nobel Prize for Medicine in 1964. It became clear to me that yeast is an ideal model organism, easy and cheap to grow in sufficient amounts for biochemical studies. However, pulse-chase experiments showed that this compound was not a precursor in mannan biosynthesis, but rather a degradation product of phytosphingolipids. Fortunately, I did not give up right away, but instead reconsidered my hypothesis. What if the lipid-bound inositol-mannoside is the precursor I looked for? Indeed, when I incubated crude yeast membranes with GDP-14C-mannose, the radioactivity rapidly appeared in the polymer fraction, the mannans. But a tiny amount—I recall 60 cpm (less than 2% of the radioactivity incorporated) running at the front of the initial paper chromatograms—was also detected in the lipid fraction. To cut a long story short, the lipid–mannose compound did not contain any inositol; however, it nevertheless became the first lipid intermediate reported to function in mannoprotein and thus in glycoprotein biosynthesis (Tanner, 1969). In subsequent years, several laboratories made similar observations in mammalian and plant cells for different sugars and eventually the ‘lipid’ component was identified as dolicholphosphate (Kauss, 1969; Behrens and Leloir, 1970; Jung and Tanner, 1973). The fact that undecaprenyl-activated sugar precursors in bacterial O-antigen and in peptidoglycan synthesis had been found a few years earlier (Higashi et al., 1967; Wright et al., 1967) positively influenced, of course, the history of the discovery of dolichol functions. We also learn from this story that wrong hypotheses can nevertheless lead to interesting and new information. I even think that the main function of a hypothesis is not to be a challenge for scientists to prove or disprove it, but rather a means to keep us scientists going. Efraim Racker once said: ‘A hypothesis is like a canoe; you only need it to cross the river’. The present discussion about hypotheses-driven and not hypotheses-driven—but potentially hypotheses-generating—research, thus, should not be taken too seriously. I believe, however, that it is more appealing to have a defined conception and to follow it with curiosity every morning when you enter the lab. Further progress in studies of protein N-glycosylation was determined mainly by work with animal cells. Dol-PP-oligosaccharide was first described in Leloir's laboratory (Behrens et al., 1971); the final structure of Dol-PP-GlcNAc2 Man9Glc3 was elucidated by Kornfeld and colleagues (Kornfeld and Kornfeld, 1976; Li et al., 1978)—strongly influenced by Clint Ballou's manno-oligosacharide structures of yeast mannoproteins (Nakajima and Ballou, 1974)—and the dolichol cycle was formulated mainly by two groups (Robbins et al., 1977; Kornfeld et al., 1978). That this complex sequence of reactions, which takes place in the ER, was conserved from yeast to humans was indicated by early in vitro results obtained by Ludwig Lehle (Lehle and Tanner, 1975; Lehle, 1980). Decisive progress concerning the molecular biology and genetics in the field of N-glycosylation came from the ingenious mutant selection work of Huffacker and Robbins (1983). Whereas in mammals a large number of different sugars can be bound to protein hydroxyamino acids (protein O-glycosylation), in yeast, only mannose was found to be linked to serine or threonine (Sentandreu and Northcote, 1969). This type of protein modification, which had long been considered to pertain exclusively to fungal cells, represented a main research topic over many years in my Institute at the University of Regensburg. Our interest in this protein modification started with the finding that Dol-P-mannose is the mannosyl donor for the first mannose that becomes O-linked to proteins in yeast (Babczinski and Tanner; 1973; Orlean, 1990). This differed completely from the many O-glycosylation reactions described in higher eukaryotes, where the sugars are transferred from sugar nucleotides. Furthermore, the yeast-type O-mannosylation starts in the ER (Haselbeck and Tanner, 1983), whereas most of the other O-linked saccharides become attached to proteins in the Golgi compartment. The extension of the O-linked mannose to the oligomannoside takes place in the yeast Golgi (Figure 1). Protein O-mannosylation in yeast A major scientific breakthrough came when Sabine Strahl purified the ER membrane protein Dol-P-Man: protein-O-mannosyltransferase (Pmt1p) to such an extent that it was possible to generate an inhibitory antibody. Using this antibody she obtained pure, although enzymatically inactive, immunopositive protein, enough to be used for N-terminal sequencing. The first few amino acids of the sequence became known during Sabine's PhD party, at the end of which my co-workers fortunately had been hiding my car key and ordered a taxi. Soon thereafter the PMT1 gene was cloned, and somewhat later the whole Pmt family, with seven members, became known (Strahl-Bolsinger et al., 1993, 1999). Whereas it was expected that PMT genes were also present in other fungi, it came as a surprise when a PMT gene was reported from Drosophila (Martin-Blanco and Garcia-Ballido, 1996). Jurado and co-workers, as well as Tobias Willer and Sabine Strahl, when checking genomic libraries of human and mouse, also found PMT genes in mammals (Jurado et al., 1999; Willer et al., 2002, 2003). However, in the higher eukaryotes only two genes were found to be present, each belonging to a specific subfamily (Figure 2). Subfamilies of protein O-mannosyl transferases. For details, see Strahl et al. (1999) During the following years, it was revealed that in yeast as well as in humans, the Pmt proteins form heteromers or homomers and are only active as these oligomers (Girrbach and Strahl, 2003; Manya et al., 2004). It was shown that different Pmt proteins have different substrate specificity for the proteins to become O-mannosylated, and that they are seven helix transmembrane proteins with the N-terminus located at the cytosolic side, and with the C-terminus facing the ER lumen (Gentzsch and Tanner, 1997; Strahl-Bolsinger and Scheinost, 1999). Finally, it was demonstrated that the activity of Pmt proteins is essential for yeast (Gentzsch and Tanner, 1996) and mouse. Knock-out mice are dying as embryos when they are 8 days old (Willer et al., 2004). For a biologist working for more than 30 years on protein glycosylation and carrying out a lot of detailed biochemical studies, it was somewhat dissatisfying not to see a general functional principle of the various sugars attached to proteins. Hakomori called it ‘an enigma’. Finding that N-linked high mannose chains are involved in lysosomal protein targeting in mammalian cells (not, however, in the corresponding vacuolar targeting in yeast), and discovering that leukocytes, through specific carbohydrate-binding cell-adhesion molecules, the selectins, interact with endothelial cells, a prerequisite for the leukocytes to arrive at the loci of infection and inflammation (see textbooks of cell biology!) seemed anectodal, considering the enormous amount of various glycosylated proteins. In the last few years, however, it has become clear that a number of very severe congenital diseases are related to underglycosylation of N- and O-linked glycoproteins. A defined molecular mechanism causing these diseases has been suggested for yeast-type O-linked carbohydrate chains (Barresi and Campbell, 2006). For the congenital underglycosylation of N-glycosylated proteins—of which up to now 19 subtypes as far as the genes affected are known—causal relations between genotype and phenotype are not really understood (reviewed in Lehle et al., 2006). The fact that congenital underglycosylation [congenital disorders of glycosylation (CDG) patients] leads to very severe children diseases—one-third of the children die before age 3 years—has made it very clear that protein glycosylation is of crucial biological importance, especially in the embryonic and post-embryonic development. In the case of a defect in the yeast-type O-mannosylation, the major phenotype is muscular dystrophy. The phenomenon of brain malformation in some of the patients indicates that the protein-bound sugars are also important for neural cell migration, and thus for structuring the brain. In all respects, yeast has contributed enormously to uncovering these various types of human disease, as has been summarized in a recent review (Lehle et al., 2006). Although I have never considered it scientifically important whether the results of basic research contribute to solutions of medical problems or, for that matter, are applied in any practical way, it was nevertheless gratifying to see that a colleague's comment, which he made about 15 years ago after a lecture of mine, did not become substantiated. He said: ‘I could never understand why an intelligent person would work all his life with these boring carbohydrates, which seem not to have any interesting function whatsoever’. They obviously have, and it is still fascinating to find out the molecular mechanisms. Naturally the shortest interval in any lifespan is the present. I consider the excursion into a new group of cell wall proteins as my present interest (although this is not fully true; see ‘The future’, below). These studies have more or less reached their endpoint with a paper published this year (Ecker et al., 2006). In the hope of finding functional roles of carbohydrates attached to proteins, we studied for some years various cell wall proteins, such as the mating-type agglutinins. During these investigations, Vladimir Mrsa, a postdoctoral co-worker from Zagreb, discovered a new group of cell wall proteins that could be released by mild alkaline treatment from SDS-extracted cell walls (Mrsa et al., 1997). In this way he identified four covalently linked cell wall proteins (Ccw5, Ccw6, Ccw7 and Ccw8), which were all highly glycosylated and none of which contained the typical C-terminal GPI anchor attachment sequence. This was the only pathway known at the time for covalent attachment of proteins to cell walls (Smits et al., 1999). This group of new, mild alkali-releasable cell wall proteins contained specific repeats of 12 amino acids, giving rise to the name ‘Pir proteins’ (proteins with internal repeats), a name originally proposed based on gene analyses only (Toh-e et al., 1993). The Pir proteins were recently found to be attached via their repeating units by an unusual ester linkage between a specific glutamic acid and glucan (see Table 1 and Ecker et al., 2006). During my postdoctoral stay with Otto Kandler in Munich (1964–1969), I started another line of research. Already in the 1950s, Kandler had observed that glucose is photo-assimilated by Chlorella under anaerobic conditions, which was later considered to be the first in vivo evidence for the existence of photophosphorylation (Arnon, 1956). We addressed the question of whether photosystem I or II, or both, were responsible for this light-dependent process, and in these experiments I noticed a certain lag period before the glucose disappeared from the medium. The study of this ‘adaptive’ phenomenon led eventually to the characterization of an inducible, active glucose transport system (Tanner, 1969). Research in the transport field was just as fascinating as research in the field of protein glycosylation, and this was the reason why I followed this topic also throughout my scientific life (Tanner, 2000). From discovering proton symport in eukaryotes (Komor, 1973), it led to the molecular cloning of the first plant uptake protein (Sauer and Tanner, 1989), if we consider unicellular Chlorella a plant, which—just to remind the reader—was the organism used for establishing the basics in plant photosynthesis. When the field of membrane transport proteins exploded (Tanner and Caspari, 1996; Rentsch et al., 1998; Büttner and Sauer, 2000), a visiting scientist from the Czech Academy of Sciences in Prague, Mirka Opekarová, proposed that we should focus our studies on the role of lipids in membrane transport, instead of worrying about more and more transport proteins. And exactly this became a new research area that determines my future activities, as long as they are still supported by grants. What is so interesting about membrane lipids? Lipids represent about half the components of biological membranes. Hundreds of different molecular lipid species are known, although a few lipid species would suffice to build up a barrier for water-soluble compounds between two biological compartments, which is considered to be the main function of membrane lipids. Their multitude thus indicates that membrane lipids must have additional functions. In the last 20 years, the role of membrane lipids in signalling has been discovered (e.g. phosphatidyl inositols; see textbooks!). Chen and Wilson (1984) were the first to report a specific lipid requirement for a transport protein. They showed that phosphatidyl ethanolamine (PE) is necessary for a fully functional lac-pemease in Escherichia coli. This effect was subsequently studied in depth by Kaback and by Dowhan (Bogdanov et al., 1996; Xie et al., 2006). We showed that transport processes in yeast are also dependent on the presence of PE. While several proton-cotransporters (Can1p, Fur4p, Mal6p) were affected in their delivery to the plasma membrane in PE-depleted cells, the facilitators transporting glucose were not affected (Opekarová et al., 2002). It subsequently turned out that Can1p and Fur4p cotransporters behaved like ‘raft proteins’, as judged from their detergent solubility (Malinská et al., 2003; Hearn et al., 2003). Confocal microscopy of Can1-GFP and Fur4-GFP revealed an interesting patchy distribution within the plasma membrane (Figure 3). Can1–GFP expressed in S. cerevisiae. (A) Confocal cross-section. (B) Surface view. For details, see Malinska et al. (2003) Thus, without intending it, we became involved in the most progressive, trendy and controversial research field of ‘lipid rafts’ (Simons and Ikonen, 1997; Bagnat et al., 2000; Munro, 2003). In our studies we showed that the plasma membrane of yeast cells is compartmented into at least two lateral subcompartments: one consists of about 50 patches, 300 nm in diameter, called the membrane compartment of Can1p (MCC); the other looks like an extended meshwork and contains the H+-ATPase, and was called the membrane compartment of Pma1p (MCP). The two compartments do not overlap. In addition to Can1p, the MCC contains Fur4p, Tat2p, Sur7p and, when heterologously expressed in yeast, Chlorella HUP1 (Malinská et al., 2003; 2004; Grossmann et al., 2006). In the MCP compartment, besides Pma1p, only Pmp2p and possibly Mid2p have been visualized so far. We also demonstrated that the amount of ergosterol, the main lipid component of the yeast plasma membrane, is significantly enriched in the MCC compartment. Finally, we showed that the presence of some of the components in the MCC patches, such as Can1p, Fur4p, Tat2p and HUP1, but not Sur7p, is dependent on the membrane potential in a reversible manner (Grossmann et al., 2007). Is the plasma membrane compartmentation really driven by a phase separation of lipids—as assumed in the classical lipid raft concept—or are mainly protein–protein interactions responsible for the membrane pattern we observe? How are the compartments stabilized in the membrane? An individual patch, for example, resides at the identical position for 30 and more minutes. Are the MCC patches really related to the recently described eisosomes, which were proposed to be involved in endocytosis (Walther et al., 2006) The main question, however, concerns the functional relevance of the observed compartmentation. I am deeply grateful to all the people who worked with me and for me in all these years. Those who are mentioned in the list of references are only a few of them. I am grateful to Dr Opekarová for helpful discussions as well as for Figure 3, and to the Deutsche Forschungsgemeinschaft for the almost 40 years of continuous financial support. Thanks for financial support also goes to the Fonds der Chemischen Industrie.