55 years together—our life with yeasts

Abstract

The authors look back at their life together in yeast research, the influences that shaped it, certain challenges and changes in laboratory and funding policies.

We met at the Faculty of Chemistry in the University of Madrid when we were less than 20 years old. Since that moment we have been together, first at the Faculty, then in the research laboratory and finally at home when we married 50 years ago. Therefore, our scientific journey and reflections will be dealt with in a common text.

JUANA MARIA—CHEMISTRY WAS A GOOD CHOICE

My interest in science started in the family. The house of my grandparents formed part of a small factory that produced soap, candles and glycerol. As a child, I sometimes overnighted with them and, occasionally in the late afternoon, I saw a lamp glowing in a small building in the back yard and was told ‘the chemist is there’. I was then allowed to go down and look at his work. I was fascinated by the manipulations. A few years later, I read a biography of Madame Curie and, at 11, I had already decided that I would be a chemist and make ‘discoveries’. It did not occur to me that being a girl would put me at any disadvantage and, in fact, at the French School in Madrid where I studied, I did as well as the best of the boys. In the preparatory year before entering the University, our biology classes were taught by a young and passionate teacher, Antonio García-Bellido (who would later become a world recognized developmental biologist). He tried to convince me that chemistry was just an ancillary science and that biology, or physics, were the real thing. I was not persuaded and started in the Faculty of Chemistry where I met Carlos.

CARLOS—A FEW BOOKS PAVED THE WAY

I became interested in science by reading biographies: quite early, one of Thomas A. Edison that was among the couple of books available in a home severely affected by the Spanish Civil War and its aftermath, then several others in a book entitled Heroes of Progress, and finally that of Ramón y Cajal, the scientist who changed neuroscience, and that of Marie Curie written by her daughter Eve. These books, acquired with difficulties in a home that was poor, but interested in human progress, were my guides to scientific life. Also during my initial years at the University, I read the ‘Rules and Advices on Scientific Investigation’ by Ramón y Cajal (Fig. 1, left). This book, now somewhat outdated in parts of its outlook but still glowing with enthusiasm for scientific research, definitively directed me towards laboratory research. I entered the University to study chemistry. At the beginning of my second year, I met Juana María, and our paths have been common ever since.

THE SERENDIPITOUS ENCOUNTER WITH ALBERTO SOLS

We both specialized in organic chemistry and gratefully remember an intensive practical course in which we got a real feeling for what it meant to work seriously in a laboratory. The basis of that course was preparations of organic compounds described in ‘the Gattermann’, a book (Ludwig Gattermann, Practicals of Organic Chemistry) whose Spanish translation was out of print even then. Carlos was able to buy a used copy that we preserved until recently when we passed it on to one of our outstanding students. It is interesting to note that Primo Levi in his book ‘La ricerca delle radici’ (The Search for Roots) places ‘the Gattermann’ among those books that were most influential for him. He writes that in it ‘one feels something that is more elevated than the mere technical information: the authority of someone who teaches things because he knows them and he knows because he has experienced them, a sober but firm call to responsibility…’ (translation by Carlos). Within that specialty, we took a course in Biochemistry that was quite boring. Even though it was 1962, we were told nothing of the revolution occurring in biology as a consequence of the elucidation of the structure of DNA. We heard about it only a couple of years later on meeting Carlos Asensio in the laboratory of Alberto Sols. Scant research was being performed in the University at that time and original papers were not given to students to read or discuss. Fortunately, over the years this has changed. We also knew little about what was going on in laboratories inside and outside the University. However, we had the great luck of having an enthusiastic professor of Chemical Engineering, Enrique Costa, who, when asked about the possibility of doing summer work in a laboratory, directed Carlos to Alberto Sols; this also led in time to Juana María joining his team.

INITIATION TO RESEARCH—FUNGAL OXIDASES

We began to work in 1963 within Alberto Sols’ group with Carlos Asensio, who had recently arrived from New York where he had worked with Bernard L. Horecker. Asensio was spreading exciting news about molecular biology, a radically new concept for us. He also shared interesting narratives of his stay in the states, where he participated in various courses, among them Van Niel´s microbiology course at the Hopkins Marine Station. In Horecker´s laboratory, Asensio demonstrated that oxidation of galactose by galactose oxidase produced the 1-6 dialdehyde of galactose, an outcome quite different from that of glucose oxidation by glucose oxidase which yielded gluconic acid. The dialdehyde seemed a curious intermediate and Asensio thought that it could be the precursor of an uronic acid. Therefore, we started exploring this possibility and simultaneously initiated a broad screen of fungi trying to detect extracellular sugar oxidases beyond the known glucose or galactose oxidases. We dedicated much time and effort to this screening, but to no avail, no new oxidase was detected. We were more successful, however, in the search for uronic acids and found that crude extracts of the fungus Polyporus circinatus (later renamed Dactylium dendroides) indeed converted galactose dialdehyde to galacturonic acid. This work led us to identify uronic acids in fungal cell walls, a finding that, to our great satisfaction, was recognized in a chapter of an Annual Review of Biochemistry. We have vivid memories of this work, the generous use of sulfuric acid and the broad palette of colors generated by Dische’s assays of different carbohydrates and derivatives.

INTO THE WORLD OF YEASTS

After some time in this line of work, Carlos started working with yeasts, under Sols’ direction, initially studying fructose-1,6-diphosphatase (now bisphosphatase) from Saccharomyces cerevisiae. Our finding that AMP inhibited this enzyme turned out to be a nice example of the need to perform enzyme assays at physiological pH, a pervasive idea in Sols’ research. Assays at alkaline pH, considered ‘optimal’ for this enzyme, had precluded detection of the AMP inhibition by groups working with another yeast. Carbon source-dependent variations in enzyme levels initiated our interest in catabolite repression that would occupy us years later. The work on fructose bisphosphatase (FbPase) was presented at an international meeting in Lisbon where we had the opportunity to meet, among other scientists, Sir Hans Krebs (Fig. 1 right); this made us feel that we really did belong to the biochemical community.

We married on 1 July 1966 and came back to the lab a couple of days later to finish preparing to defend our Ph.D. theses, scheduled 10 days later. Afterwards, Juana María joined Sols studying different aspects of yeast biochemistry. During this period, we studied the ‘feed-forward’ activation of pyruvate kinase by fructose-1,6-diphosphate (now bisphosphate) discovered by Benno Hess (Dortmund). Sols was surprised by this ‘feed-forward’ regulation because it did not fit the feedback paradigm of regulation that dominated the regulatory landscape. ‘Feed-forward’ had a suspicious ‘teleonomic’ smell, so he asked us to put aside our work and perform a series of assays to check these observations. Our results showed that Hess was right but that in certain yeasts pyruvate kinase showed a different kind of regulation mediated by carbon source-dependent changes in enzyme levels. Our last work in Sols’ laboratory characterized enzymes in glycerol metabolism and developed a model for the structural requirements of glycerokinase substrates. The paper dealing with this work was written after we moved to Germany to begin postdoctoral work. Our first purchase there was a typewriter to type the manuscript.

THE SPIRIT OF VELAZQUEZ

Working with Sols played a decisive role in our scientific lives. Sols was one of the discoverers of hepatic glucokinase, its regulation by insulin and of an allosteric effector of mammalian hexokinase. He also made important contributions to our understanding of phosphofructokinase (PFK) regulation. Sols was an exceptional person, a great scientist with a wide curiosity, experimental rigor and exemplary dedication to his work. His insistency in avoiding botchery and mediocrity permeated his students. He has been the person who exerted the deepest influence on our scientific life. His great care in presenting results either in an article, a conference or a group seminar was enormously important in our apprentice years. The process of writing manuscripts under his guidance and constructive criticism was a piece of teaching that remains one of our most important debts to him. He was also remarkably generous with his scientific ideas and his ability to accept criticism of his opinions, even when coming from beginners. He was a towering figure at a time when high-quality biochemical research was not widespread in Spain.

Another important influence on us was the intense communication with other groups of the, then small, biochemical community working in the same building as Sols. The hard work and commitment to high standards of the Julio Rodríguez Villanueva and Manuel Losada research groups were an example for interested beginners. We have seldom found such an atmosphere of scientific interest, open discussion, spirit of mission and friendship. Asensio named it ‘the spirit of Velázquez’ making a play of words with the name of the Velázquez-street where the laboratories were located and the artistic essence of the great painter.

HELMUT HOLZER’S LABORATORY—SCIENCE, POLITICS AND WALKS IN THE SCHLOSSBERG

After 4 years in Sols’ group, we went as post docs to Helmut Holzer's laboratory in Freiburg im Breisgau, a small German university town in the Black Forest, where we spent two memorable years (1967–1968). Holzer had elucidated the structure of ‘active acetaldehyde’ in the late 1950s and had become interested in the regulation of enzyme activity. His finding of glutamine synthetase ‘inactivation’ in Escherichia coli launched his interest in reversible covalent modification of enzymes and its regulatory role. Later on, this type of regulation became important enough to elicit a series of periodical meetings (Metabolic interconversion of enzymes) with scientists such as Earl R. Stadtman, Edmond H. Fischer, Edwin G. Krebs, Shmuel Shaltiel, Alberto Sols, Helmut Holzer, Henry G. Hers, actively participating in them. Years later, we were lucky to attend these meetings and to participate in organizing one of them.

Following Holzer's policy regarding couples, it was decided that Carlos would work in a group studying E. coli glutamine synthetase, while Juana María worked on yeast, one floor upstairs, with Wolfgang Duntze. Holzer came every week to our labs to assess our progress and make suggestions. Mentoring by Holzer was important for us because he provided an example of how to respect people in the lab and colleagues in the scientific community. At the personal level, we maintained a respectful friendship with Holzer and his wife, Erika, which continued through visits and correspondence until their passing away.

Holzer had found that addition of ammonium to an E. coli culture growing with another nitrogen source abolished assayable glutamine synthetase activity. This ‘inactivation’ was reversible and could be reproduced in vitro using crude extracts; it required a protein fraction we called ‘inactivase’. Carlos worked on purifying this enzyme with Eberhard Ebner in a stimulating collaboration. It was found in Holzer´s laboratory and in that of Stadtman in Bethesda that the inactivation was caused by a reversible adenylylation. The Stadtman laboratory subsequently identified regulatory cascades demonstrating E. coli glutamine synthetase control to be one of the most complex known at the time. Riding the wave of protein interconversion, it appeared interesting to investigate whether the two forms of yeast PFK, with differing sensitivities to ATP, described by Sols, might be due to interconvertible forms of the enzyme. Juana Maria's isolation of a heat-labile yeast fraction that made PFK insensitive to ATP inhibition certainly pointed in this direction. However, interconversion could not be demonstrated and a nice hypothesis had to be abandoned.²

Juana María also studied the regulation of the Saccharomyces cerevisiae glyoxylate cycle enzymes, especially the three isoenzymes of malate dehydrogenase. She used electrophoresis in agar gels to follow how various carbon sources influenced the levels of these isoenzymes. The system available was rather primitive, and it took a while to get the knack of it. The enzymatic activity was assessed in situ using a coupled assay that yielded an insoluble dark blue product. For a time we were puzzled by the presence of a ‘nothing dehydrogenase’, a blue band that appeared in the control lane without malate. Later on, we suspected that it was an ethanol dehydrogenase acting on the small amount of ethanol present in our reagents.

Besides the science, our stay in Freiburg was game-changing in other ways. There was a lively political atmosphere, very different from that found in Spain that was still suffering under a dictatorship. The students were very militant, Mao´s Red book was held in high esteem (we have still our old copy), and demonstrations were frequent and increased after the shooting of a student, during the 1967 Berlin visit of the Persian Shah. Shortly thereafter, the Soviet invasion of Czechoslovakia, ending the political experiment of ‘socialism with a human face’, dealt a heavy blow to many hopes. The political effervescence permeated the laboratory with lively exchanges of ideas at after-lunch coffees. While we rather sided with the students in these discussions, sympathizing with their calls for change, we were also critical of the tactics used by the extremists in decision-making assemblies. When a vote failed to support their positions, they initiated new rather hollow discussions prolonging the assemblies until tired participants started to leave. Then the rejected proposal was put to another vote and accepted by the die-hard activists who had remained. This proposal, of course, could not be reconsidered because ‘it had been democratically approved by the assembly’. Years later, we experienced similar manipulative behavior in Spain by individuals who pretended to be progressive and democratic but were not.

At the same time, we enjoyed a rich cultural life. We discovered artists such as Franz Marc, Vasily Kandinsky, Lyonel Feininger or Käthe Kollwitz, went to see classic films such as The Battleship Potemkin, Les enfants du Paradis, Der Zerbrochene Krug, and had access to new literary works including those of East European authors. The surroundings of Freiburg allowed us extended walks in the forests around the Schlossberg. We delighted in the color changes as one season of the year transitioned into the next. Although our personal life was rather austere, saving money that would be needed on our return to Spain, the two years in Freiburg remain a prominent landmark in our memories.

START OF INDEPENDENT RESEARCH

Back in Madrid, we found Sols’ department transformed into the new Institute of Enzymology, though no more space or new instruments were allocated to it. At that time, the institute was almost completely dedicated to the organization of the 1969 meeting of the Federation of European Biochemical Societies (FEBS) in Madrid. This was an important event for the Spanish Society of Biochemistry—itself owing a great deal to Sols activity—since it was its first big international adventure. Unfortunately, the country's political situation almost led to the failure of that venture. Student and social unrest led the government to decree a 3-month state of emergency that overlapped with the date of the meeting. This decree abolished the citizens’ few legal guarantees and allowed police to enter the university. Consequently, several European scientists initiated a movement to boycott the meeting if the state of emergency continued to overlap with the meeting. Had it succeeded, this move would have been disastrous for the fledging Spanish biochemical community. The organizers, Sols at the helm, with the help of Severo Ochoa, then in New York, convinced FEBS constituent societies to abandon the strategy. Societies of the Eastern Block were, for obvious reasons, also interested in separating science and politics. The boycott was finally averted, the state of emergency revoked, with a very successful meeting being the outcome. A poster designed by Salvador Dalí (Fig. 2, left) is still treasured by many biochemists who attended the meeting.

It was in this atmosphere that we tried to initiate our first line of research. Our studies of glutamine synthetase inactivation and the publication by Jean-Marie Wiame’s group in Brussels of a similar process, the disappearance of yeast ornithine transcarbamylase activity after a change in the nitrogen source, led us to consider whether some enzymes might also be ‘inactivated’ by a change of the carbon source. A good candidate appeared to be the gluconeogenic enzyme FbPase expressed in gluconeogenic conditions, but not in the presence of glucose. We had performed some Sunday experiments, while in Freiburg, on the possible inactivation of this enzyme with results that seemed promising and decided to follow this lead. In fact, we found that addition of glucose to a culture growing on gluconeogenic carbon sources produced a rapid disappearance of the FbPase activity. Together with our first Ph.D. student, Jesús Molano, we strenuously tried to reproduce the inactivation in vitro with consistently negative results. Later on, we demonstrated, with him and a Brazilian student, Shigehiro Funayama, that in vivo FbPase inactivation was due to proteolysis. Meanwhile, Holzer’s laboratory reported a biphasic inactivation: a very rapid initial 2-fold decrease in activity followed by a slower one. With María-Jesús Mazón, a new addition to our group, we showed that the rapid inactivation was due to phosphorylation by a cAMP-dependent protein kinase, leading us to conclude that phosphorylation tagged FbPase for proteolytic degradation.

In his posthumously published autobiographic notes, Sols regretted excessive dispersion in the subjects he tackled, writing that ‘I often got distracted shooting at any small fry that came across my way’. At times, our curiosity also led us to follow too many sideways. Here we will consider only some of the topics in which we have been interested. Sols had been concerned about the differences between conditions in which enzymes work in their cellular environment and those we impose on them in vitro, diluting proteins up to 1000-fold thereby eliminating their interactions with other proteins. During a sabbatical visit, Richard E. Reeves (Louisiana State University) developed a method to permeabilize E. coli which allowed us to assay enzymes ‘in situ’. With our colleague Ramón Serrano, we developed a similar approach for yeast and studied the regulation of glycolytic enzymes. A new student, Marcelino Bañuelos, examined the functioning of the complete pathway finding that the K_m, V_max and regulatory properties of various enzymes were the same in conventional extracts as in situ. The in situ rate of fermentation, however, was 2-fold higher than in vitro and the lag time before active fermentation began was much shorter. During this study, we established a friendship with Pierre Labbe and Rosine Labbe-Bois in Paris who were also interested in this methodology.

ADDING STRINGS TO OUR BOW

In the mid-1970s, we recognized that our enzymological toolkit was insufficient and that new approaches would be necessary to further understand in vivo regulation. During a 1972 FEBS-ICRO course on ‘Enzyme Regulation’ that Sols organized, we met Dan G. Fraenkel, who invited us to visit at his Boston laboratory. We gladly accepted and each of us remained there for 6 months with an overlap of 2 weeks. There we participated in the initial attempts to establish connections between yeast genetics and glycolysis which influenced our subsequent work; we recognize Dan as an outstanding researcher and are delighted to continue being his friends. During our stay at Harvard University, the societal implications of the new biological research were front line. The Asilomar Conference on the use of recombinant DNA technology had recently taken place and ‘Science for the People’, an organization where Jon Beckwith was a central figure, tried to mobilize scientists to put people first.

Our first steps in yeast genetics were invaluably assisted by Jaime Conde, a yeast geneticist from Seville, who introduced us to the intricacies of spore micromanipulation—with self-constructed needles—and to all the pleasures and puzzles of Mendelian segregation, gene conversion and the like. These spore analyses occasionally show unexpected marvels (Fig. 3). It also became important to acquire skills with recombinant DNA techniques, and for this Juana María spent some time in the laboratory of François Lacroute in Strasbourg. This was a great help in transitioning from enzymology to molecular biology, a task for which we want to highlight the efficient contribution of our students, Raquel de la Guerra and Marta D. Valdés-Hevia.

ESCHERICHIA COLI AND SACCHAROMYCES CEREVISIAE DO THE SAME THING DIFFERENTLY

We had shown that FbPase is repressed in the presence of glucose; this characteristic, shared by many genes involved in carbon metabolism, has been called catabolite repression and is widespread among microorganisms. Since decreased intracellular cAMP was responsible for catabolite repression in E. coli, this was assumed to be also the case in S. cerevisiae, and indeed, a couple of papers appeared reporting it. Johannes B. Van der Plaat, however, made an intriguing observation; upon adding glucose to yeast cells growing in gluconeogenic conditions, the intracellular concentration of cAMP sharply increased in less than a minute and then immediately decreased, leveling off a few minutes later. Yet, whether cAMP was lower in glucose-grown yeast cells relative to other carbon sources was unanswered.

Using a more robust methodology than in the initial reports, we demonstrated that catabolite repression in yeasts was not associated with low cAMP (Eraso and Gancedo 1984). Clearly, a ubiquitous regulatory molecule, such as cAMP, could act in different ways in different organisms. Actually, years later with Chris Lindley and Oscar Zaragoza, we showed that increased cAMP was sufficient to repress FBP1 transcription, but not that of SUC2, another gene subject to catabolite repression. More recently, we demonstrated with Jack Pronk's group in Delft that catabolite repression of FBP1 and other genes occurs in mutants lacking cAMP-dependent protein kinase, indicating the redundancy of regulatory mechanisms. We also identified FBP1 promoter elements responsible for its transcriptional regulation; Juan José Mercado, Olivier Vincent, Joelma F. de Mesquita and Oscar Zaragoza participated in these studies through the years.

REGULATION GENERATES A SLIGHT BUT SIGNIFICANT ADVANTAGE

Interested in the physiological importance of regulatory mechanisms, we investigated mutants with defects in FbPase control. Klaas van de Poll in Utrecht and Julius Marmur in New York had isolated mutants unable to inactivate FbPase, fdp and cif, respectively. These mutants failed to grow in glucose reportedly due to a futile cycle, i.e. simultaneous operation of FbPase and its glycolytic counterpart PFK, leading to a precipitous drop of ATP. We invalidated that explanation by showing that mutant strains constitutively expressing both FbPase and PEPcarboxykinase, another gluconeogenic enzyme subject to inactivation by glucose, were still able to grow in glucose and maintain normal ATP levels. To test whether this was due to allosteric inhibition of FbPase, we studied the behaviour of yeast strains expressing a fructose-2,6-bisphosphate and AMP-insensitive FbPase and found that even though the proportion of fructose-1,6-bisphosphate recycled to fructose-6P reached 14%, there was still no decrease in ATP levels. Nevertheless, growth in a mixed culture of a wild-type and a mutant strain, expressing unregulated FbPase on glucose, showed that FbPase regulation provided a significant, although slight, competitive advantage (Navas and Gancedo 1996).

FITTING PIECES OF THE PUZZLE TOGETHER

Studying the fdp1 and cif1 mutants mentioned above led us to discover a new regulatory mechanism for glycolysis. Van de Poll had mapped the fdp1 mutation to a region of chromosome II near LYS2. At the time we investigated the fdp1/cif1 mutants, Carlos and Horst Feldmann served as FEBS Executive Committee officers and became good friends; Carlos spent then a month in Feldmann’s Munich lab sequencing PCK1 with Rolf Stucka. Since Feldmann was sequencing chromosome II, it was ‘selbstverständlich’ that Carlos asked Feldmann to help clone the FDP1/CIF1 gene. With this collaboration, Maribel González in our lab cloned the wild-type CIF1 gene but the putatively encoded protein sequence was not similar to any known protein sequence at the time (1992). Various results subsequently led us to conclude that the encoded protein regulated glycolysis by coupling in some way the rates of its ATP-consuming and ATP-producing steps. Shortly thereafter, Andreas Wiemken and his colleagues in Bale cloned the trehalose-6-phosphate synthase gene and found its sequence to match the one we published for CIF1; we proposed therefore to rename CIF1/FDP1 as TPS1. Although this gave Tps1 a function, it did not explain why cells lacking Tps1 failed to grow in glucose, or why there was a drop of ATP and no inactivation of gluconeogenic enzymes when glucose was added to the mutant growing in a gluconeogenic carbon source. Following an idea of Juana María, Miguel A. Blázquez showed that the product of the Tps1 reaction, trehalose-6-phosphate, was a potent unknown inhibitor of yeast hexokinase and that loss of this ‘brake’ led to uncontrolled phosphorylation of glucose and concomitant depletion of ATP (Blázquez et al.1993) thereby leading to growth arrest in glucose. This inhibition is now incorporated in mathematical models of glycolysis.

An important return of this work was the cloning by Miguel Blázquez and Carmen-Lisset Flores of the gene encoding trehalose-6-phosphate synthase from Arabidopsis thaliana. This work, and the cloning of the gene encoding trehalose-6-phosphate phosphatase by Andreas Wiemken’s group, published in the same issue of Plant Physiology, demonstrated that trehalose was also present in higher plants.

PYRUVATE CARBOXYLASE—THE ELUSIVE MUTANT AND INTERNATIONAL COLLABORATION

Helping a friend, Pierre Barre (Montpellier, France), who was interested in a mutant lacking pyruvate carboxylase (Pyc), opened new perspectives. Since Pyc is an anaplerotic enzyme necessary to replenish the TCA cycle, pyc mutants should be unable to grow in minimal glucose ammonia medium. With this rationale, we searched for pyc mutants, but after several failed rounds of mutagenesis, we set the problem aside. In the meantime, ScPYC1 was cloned prompting us to determine whether our inability to isolate the mutant was due to the existence of a second gene, although in 1991 it was not common knowledge that S. cerevisiae contained many gene duplications. Again, Feldmann’s lab in the person of Rolf Stucka was helpful and probing yeast chromosomes with a PYC oligonucleotide we found two signals, in chromosomes II and VII. Disrupting both PYC genes yielded a strain with the characteristics expected in our original search strategy. We specially value this piece of work (Stucka et al.1991) because it involved classical genetic analysis, molecular biological techniques and biochemical assays, and derived from collaborative work in three different European countries: Rolf Stucka in Germany, Sylvie Dequin and Jean-Michel Salmon in France and Carlos in Spain.

The pyc double mutant also led us in a new direction. Javier Gamo and Miguel Blázquez isolated pyc suppressor mutations one of which María-José Lafuente and Javier Gamo found to be allelic with a mutation found in the lab that suppressed the toxic effect of glucose on phosphoglyceromutase mutants, and impaired glucose transport and catabolite repression. We identified the corresponding wild-type gene as MTH1 and demonstrated that Mth1 interacts with the cytoplasmic tails of the glucose sensors Snf3 and Rgt2. This interaction was affected by mutations in Mth1 and by the concentration of glucose in the medium implicating Mth1 in glucose signaling (Lafuente et al.2000).

In Yarrowia lipolytica, Carmen-Lisset Flores constructed a pyc mutant that was still able to grow in glucose medium. We proved that, in this case, the phenotype did not derive from the presence of two genes, but was due to incomplete glucose repression of the glyoxylate cycle. Additional disruption of the isocitrate lyase gene generated the ‘canonical’ no growth phenotype. This raised the question about how other proteins repressed by glucose would behave in Y. lipolytica. Together with Carmen-Lisset Flores and Raquel Jardón, we unexpectedly found that the gene encoding FbPase was not repressed by glucose. Another surprise was that mutants without FbPase were able to grow in gluconeogenic carbon sources because of the existence of another phosphatase, with a high K_m for fructose-1,6 bisphosphate, which vicariously assumed the role of the main enzyme (Jardón, Gancedo and Flores 2008).

UGLY DUCKLINGS OR SWANS?

Although most of our work has been performed with S. cerevisiae, we have been interested in other yeast species with Carlos shifting almost completely to Yarrowia. Switching to this species was initiated during our study of trehalose-6-phosphate inhibition of hexokinase and has been greatly facilitated by our friends Angel Domínguez in Salamanca and Claude Gaillardin in Grignon (France). Studies with non-conventional yeasts far from being ‘stamp collecting’ are important to understand the evolution of regulatory mechanisms and might turn some of these ugly ducklings in pretty swans.

BEYOND THE LAB

Besides our work in the laboratory, we have served as members of journal editorial boards, evaluation panels and scientific societies. Carlos has been quite active in the Spanish Society of Biochemistry and Molecular Biology and in FEBS, where he was Chairman of the Fellowships Committee for several years. Carlos taught general biochemistry to medical students for 15 years at the Faculty of Medicine of the Universidad Autónoma in Madrid, where our laboratory was located for almost 20 years. This was a very enjoyable task and the friendship of many former students is one of the greatest satisfactions from this period.

CHALLENGES AND BENEFITS OF OUR CAREERS TOGETHER

We begin with two issues: one is gender and the other one the effects of working as a married couple. Juana María had never considered that being a woman would put her at a disadvantage. In the building where we started to work, there were a number of well-respected, established women scientists doing research, among them Gertrudis de la Fuente in Sols’ group. This reinforced her idea that gender ought not to be a hurdle for a scientific career. At the time this was perhaps a rather uncommon situation, as in other places practically all the senior positions were held by men. Initially, we published together but it soon became clear that, with regard to the outside and the origin of ideas in our research, we would encounter a mixture of the gender problem and of spouses working together. Some would say ‘This is Carlos’ work, Juana María is just following suit’ and it would be difficult for Juana María to progress up the scientific ladder. Therefore, we decided to separate our paths, to some extent, thereby giving each of us a recognizable profile. Nevertheless, all our scientific work has always been thoroughly discussed together and therefore it has been presented here jointly using a collective ‘we’. This was the only difficulty we felt in our work as spouses. From a personal point of view, working together in the laboratory, discussing scientific questions or sharing other laboratory concerns has been an important element of satisfaction and stability in our life. The challenge of coordinating laboratory and family life was solved with the help of Carlos’ parents who agreed to live with us, taking care of our son and dealing with much of the household management while we were in the lab.

The initial period after our post doc was quite difficult. Carlos had a fixed appointment with a low salary and Juana María had only a 3-month contract. When it expired, we had to rely on money we had saved in Germany to make ends meet and we seriously considered going back to that country. The birth of our son coincided with a promotion for Carlos, but some unease arose in the department when Juana María took a year off. However, at the end of the year Juana María went through a selection process and obtained a permanent position that allowed us to concentrate on our work.

Scarcity of means at the lab permeated our initial years and eased only intermittently. Financial resources were meagre in Sols’ laboratory, teaching us the value of each reagent when planning an experiment. Due care of the equipment was also imprinted on us from the very beginning. The work of everyone in the group depended on a few basic instruments: a Klett colorimeter, a Beckmann DB spectrophotometer or a Warburg respirometer. Cuvettes for the spectrophotometer were shared by all and treated with utmost care; the quartz cuvettes were held in special high reverence.

Our laboratory budget was somewhat more comfortable during the 1980s and early 1990s with the possibility to access European Union funds. Unfortunately, the last decade has witnessed drastic financial deterioration. It has become very hard to finance Ph.D. students. Additionally, the Institute environment, far removed from young science students, has made it difficult to find good candidates to work in the lab. Another problem has been the progressive disappearance of technicians with routine tasks having to be assumed by students (and in difficult times by the senior researchers themselves).

SPREADING THE WORD ON YEASTS

In the late 1970s, we started organizing courses consisting of lectures and practical work emphasizing the potential of yeasts. They were influenced by what we experienced during a Bacterial Genetics course taught by Peter Starlinger and Walter Vielmetter at the University of Cologne in 1968 and in the Yeast course of Cold Spring Harbor organized by Gerald Fink and Fred Sherman that Juana María attended in 1976. Courses, initially restricted to Spanish participants, were opened later to the international community (Fig. 2, right) and lasted into the late 1990s thanks to funding by FEBS and the Fundación Juan March. We were also helped in these courses by others, Jaime Conde, Ramón Serrano, Marco A. Delgado, Isabel López Calderón, José M. Siverio and James M. Cregg. Thanks to two private institutions, the Fundación Juan March and the Fundación Ramón Areces, we also organized several international symposia on particular topics related to yeasts.

We also held different lecture courses at the Gulbenkian Foundation in Oeiras (Portugal) when Niko van Uden was in charge of the Advanced Courses Program, as well as in the group of Cecilia Leao in Braga (Portugal), and at the Centro Nacional de Investigaciones Científicas in Havana (Cuba). We maintained a sustained relationship with the Cuban biochemists and many of them spent time in our lab; in general, we admired their positive attitude at work and their solid background preparation. Our visits to Cuba also allowed Carlos to personally know members of his family who immigrated to Havana in the early decades of the past century (his father was also an immigrant there, but in the wake of the Great Depression returned to Spain).

Finally, we have dedicated significant efforts to knitting the increasing numbers of the Spanish yeast community together. This has been a successful enterprise that has fostered biennial meetings of group leaders and an open exchange of information and materials for 20 years. Jesús Plá from the Faculty of Pharmacy of the Madrid University Complutense has been a helpful partner in this activity.⁴

GRATITUDE—DON’T FORGET THE SOURCE

‘When you drink water don’t forget the source’ says a Chinese proverb reminding us of the importance of gratitude. We are indebted to many who influenced or helped us develop our work. Two books opened up new vistas for us. James Watson's ‘Molecular Biology of the Gene’ is a masterpiece of how to write with a rigorous but engaging style on a complex subject. Stanier, Doudoroff and Adelberg, in ‘The Microbial World’, used a new approach to give an integrative view of a bewildering diversity of microorganisms.

We have already mentioned several persons who were important for us, among them the leading figures of Sols, Holzer or Rodríguez-Villanueva. Some others shall be explicitly mentioned here. Rosario Lagunas, in our Department, and Fernando Moreno and Pilar Herrero, in Oviedo, have been friends with whom we shared projects, some publications and many discussions. Our long and continued contact with Hans van Dijken, Jack Pronk and the Delft group has been—and continues to be—a source of intellectual and human enrichment. We also enjoyed a fruitful collaboration with Alistair J.P. Brown (Aberdeen), and friendly interactions with Fritz Zimmermann (Darmstadt) and Karl-Dieter Entian (Frankfurt).

Our students contributed by performing experiments and providing valuable ideas. In addition to those mentioned, Klaus Schwerzmann, Manuel Hernández-Jodra, Javier Perea, Soledad López, Francisco Portillo, Jean Marie François, Cristina Rodríguez, Thomas Petit, Javier Menéndez, Trayana Nedeva, Mónica M. Belinchón and Daniela Livas helped in different ways to make our work possible.

In a time of scarce financial opportunities, the Deutscher Akademischer Austausch Dienst supported Juana María in Freiburg and later on, back in Spain, provided us with an electrophoresis power supply that we still use today! Carlos was supported in Germany by the Fundación Juan March and the Freiburg University. Julio R. Villanueva at the Fundación Ramón Areces has been a source of inspiration and continuous support in organizing symposia around yeasts. Last but not least, we thank the institution in which we have worked, the Spanish Research Council (CSIC); in spite of its defects and need of reforms, it has been a relatively safe haven for our research.

VIEW FROM THE LAST WINDING OF THE ROAD

Looking back at 50 years working in the lab, we see deep changes in many aspects of scientific life. Technically, our laboratory surroundings are only vaguely reminiscent of those found when we entered research. Some changes came smoothly, integrating seamlessly into our daily routine without being noticed. Others came more abruptly with the force of an earthquake. Many precious instruments such as the Warburg respirometer or the paper chromatographic chambers are all but gone, while new ones such as thermocyclers are now a basic requisite in our lab. Our interests have also changed. In the 1970s, enzyme kinetics, Bi-Bi, ping-pong and concerted models filled our seminars and coffee/tea conversations. These terms are now archaeological curiosities to our new students. Understanding metabolism was our main goal; it remained so for us, but many people found it old fashioned. Only recently has interest in this area revived.

Paper libraries have disappeared and we grieved as valuable journal collections and books were thrown away. The Pasteurian ‘paix des bibliothèques et laboratoires’ (peace of libraries and laboratories) has been lost, although whether the peace of laboratories has ever existed is a question to ponder. We no longer experience the pleasure of going to the library to browse newly arrived journals or look to back issues, finding serendipitously some interesting article.

When we started our scientific life, funding was hard to find but it could be obtained for curiosity-driven research. The more recent trend seems to allocate most research funds to problems felt by society to be ‘relevant’ or to those expected to yield short-term economic rewards. To use Baconian words, experimenta lucifera are being pushed back to give preference to experimenta fructifera, ignoring the historical records of fruitful applications derived from experimenta lucifera. To ask for a rigid timetable of deliverables or milestones, even in projects of applied nature appears to ignore basic tenets of science. As Sols wrote more than 50 years ago ‘… tomorrow´s results may destroy the plans of today. It is not possible to design a road in an unexplored land’. These words challenge the expectation of rigid adherence to the programs described in grant applications for basic and other types of research. Perhaps the availability of money to carry on ‘pure research’ that we experienced in the 20th century was an anomaly in history. Funding for research has not grown proportionally with the number of scientists devoted to it. The resulting mismatch is a matter of concern especially for the young. It is clear, however, that exponential growth cannot proceed indefinitely (Gancedo 2010) and that this problem needs urgent consideration from scientific and societal bodies.

AT THE CLOSE

At this writing, Carlos continues working in the laboratory collaborating with Carmen-Lisset Flores who has for years been a driving force in the lab. One of the current interests of the lab is to investigate the occurrence and significance of moonlighting proteins in yeasts (Flores and Gancedo 2011). Juana María has left the bench but continues to follow the literature and to write (Fig. 4). Along our journey, we have tried to adhere to a set of values that we considered important and that we find mentioned as such in the First Presidential address of Barack Obama: ‘…honesty and hard work, courage and fair play, tolerance and curiosity, loyalty…’. If we made mistakes, they were, as William Osler said in a farewell address, ‘mistakes of the head not of the heart’.

Acknowledgements

We thank the diverse funding agencies that made possible our work. We are grateful to Terrance G. Cooper for his thoughtful suggestions and careful editing. The help of Javier Pérez and Silvia Cuena in the elaboration of the illustrations is gratefully acknowledged.

Conflicts of interest. None declared.

REFERENCES