The Nobel Prize in Chemistry 2004
The interaction with Irwin Rose
As noted, I spent an important part of my graduate studies in Ernie’s laboratory. Avram spent a sabbatical in his laboratory in 1977-1978, and I joined him for the first time for several months in the summer of 1978, after I completed the initial characterization of APF-1 in Haifa. I returned to Ernie’s laboratory during the summers of 1979, 1980, and 1981. As noted, during our summer stay in 1979, we resolved the problem of the nature of the high molecular mass “compound” generated when APF-1 was incubated with Fraction II in the presence of ATP. This change in the molecular mass of APF-1 was discovered several months earlier in Haifa. However, we were not able to unravel the nature of the “compound”; this had to await the knowledge and wisdom of Ernie. In a breakthrough discovery, we found that the target substrate is covalently modified by multiple moieties of APF-1, a reversible modification that renders the protein substrate susceptible to degradation. This was a novel type of post-translational modification (see, however, above for the modification of histone H2A by ubiquitin) and clearly a new biological paradigm, that the elucidation of which required – as I feel today in retrospect – a different type of knowledge in biology and enzymology, and an original experimental approach. Elucidation of this modification would not have been possible without Ernie’s advice that was based on his immense knowledge in enzymology and protein chemistry, accompanied by his unbiased original thinking and approach to problem resolving. This discovery, along with the discovery in 1980 that APF-1 is ubiquitin, made Ernie and his fellows critically important partners in the historical trail of the discovery of the ubiquitin system. Interestingly, Ernie studied proteolysis before Avram joined him first, but had never published in the field before.
Post-graduate training at MIT and how I continued my studies on the ubiquitin system independently
The five years in graduate school had a significant impact on my future career. Not only because I played an active part in the discovery of such an important pathway, but maybe more important, because I learnt several basic and key principles on how to approach a scientific problem. From my mentors I learnt two principles: first, to select an important biological problem, preferably an unobvious one and not in the mainstream, and second, to make sure that there are appropriate research tools to approach it experimentally. I also learnt to become a long books author rather than a short story writer: I learnt not to be opportunistic but rather to adhere to a project, to dig deeply into a problem, to resolve it mechanistically, to unravel complex mazes – peeling them like an onion, not to be tempted to be dragged after fashions. I learnt to pay attention to small details, to carefully examine hints, as the important findings are not always obvious from the apparent results. I learnt to be stubborn, to fight difficulties uphill, but most importantly, to be critical: I believe I developed good senses that enable me to distinguish false from truth, and artifacts from meaningful findings. Interestingly, I learnt all these principles not in frontal lessons or formal presentations, but as an apprentice, following my mentors’ own attitude and way of thinking. At the same time I also learnt to question, to doubt, to ask, and to discuss, to follow my own gut feeling when it was necessary, not to always take advice and direction for granted, and to trust myself too. It did help in many occasions along the way. Thus, at times I found myself swimming alone against the stream. Altogether, these principles generated an important philosophy and shaped my approach to science, something I try to instill in my own students, as I strongly believe it is the only way one can make an impact, leave an imprint behind.
Towards graduation I had to think of the next step – post-doctoral training and planning of my future career as an independent scientist. I was in a dilemma. On the one hand, I knew it was important to obtain training somewhere else, under different mentorship, in a different environment, being exposed to a different culture of science. On the other hand, I knew for certain that the ubiquitin system was extremely important and that we were seeing only the tip of its iceberg. I therefore wanted to continue my studies in a related field, learning more on regulated proteolysis, but also to continue my own studies on ubiquitin. I had several ideas in mind on where to go, but the choice was quite narrow and also risky, as I did not have any idea of how much independence I could have as a post-doctoral fellow. Searching for a mentor, and with the advice of my colleague Mickey Fry, I looked for scientists whose work was related to regulated proteolysis. I wrote to Günter Blobel in the Rockefeller University, who worked at that time on translocation of proteins across the endoplasmic reticulum (ER) membrane, a process that involves cleavage of the leader peptide by signal peptidase, to Jeffrey Roberts in Cornell University who worked on E. coli RecA protein-directed cleavage of phage repressor and its requirement for polynucleotide, and to Harvey Lodish at the M.I.T. who worked, among other subjects, on processing of viral polyproteins. I am not sure Harvey was that impressed with the ubiquitin system at that time, but he was the only one to respond positively. Typical of his etiquette (as I learnt later), his response was prompt and direct, and he invited me for an interview after which he accepted me. Günter was kind enough to let me know he did not have space in his laboratory at that time, and Jeffrey never responded.
With two fellowships, one from the Leukemia Society of America and one from the Israel Cancer Research Fund, ICRF, I started a period of three wonderful years (1981-1984) in Harvey’s laboratory in the Department of Biology at M.I.T. Harvey gave me complete freedom to choose my research subjects. What I had in mind was to take advantage of the exceptional strength of the laboratory and Harvey’s unique expertise in cell biology, but in parallel, to continue my own studies on the ubiquitin system. I realized that Harvey was no longer interested in viral proteins processing, and along with Alan Schwartz who was a visiting scientist (from Harvard Medical School) in the laboratory, we started to characterize the transferrin receptor on a human hepatoma cell line with the aim of disseding the dynamics of the receptor and iron delivery into cells. This collaboration led us, along with another fellow in the laboratory, Alice Dautry-Varsat (from the Pasteur Institute) who joined us later, to the discovery of a fascinating mechanism of how iron is delivered into cells, a process mediated by the transferrin receptor: In the neutral pH of the growth medium, the iron-loaded holo-transferrin binds to its receptor with a high affinity and is endocytosed into the cell. At the low endosomal pH, the affinity between iron and transferrin is weakened dramatically. As a result, the iron cation is released, but the apo-transferrin, which has high affinity for the receptor at acidic pH, remains bound strongly. Along with the receptor, the apo-transferrin recycles to the cell surface. At the neutral pH of the growth medium, the apo-transferrin loses its high affinity to the receptor and is released into the neutral pH extracellular fluid. There, it binds iron with high affinity, rebinds to its receptor and the hololigand is endocytosed again. The transferrin-transferrin receptor pH- and iron loadingdependent cycle has become a “classic” in the field of receptor mediatedendocytosis. Based on this mechanism, other phenomena related to receptor and ligand recycling to the cell surface or targeting to the lysosome could be explained, which are also due to the pH difference between the external environment and the interior of the endocytic pathway vesicles. However, throughout this time, I lived under the strong feeling that the ubiquitin system had barely started to emerge, with only the basic principles unraveled: I felt compelled to get back and work on it. So gradually I started to “crawl” and return to my alma mater’s research subject.
On one fascinating subject I worked on my own, continuing to explore a mysterious finding I discovered during my graduate training and which I did not pursue at the time: When we purified APF-1/ubiquitin in Haifa, we noticed a large discrepancy between its dry weight and its Lowry or 280 nm protein quantitative measurements. We hypothesized that the protein can be a ribonucleoprotein, RNP, and the remaining mass is that of the nucleic acid component. To test this hypothesis, I added DNase to the crude extract in which we monitored ATP- and ubiquitin-dependent degradation of BSA, that was used as one of our model substrates. The enzyme had no effect. We then added RNase A, and to our surprise proteolysis was completely inhibited, even with an extremely small amount – mere few nanograms – of the enzyme added: it looked as if the enzyme exerted its effect via catalysis – RNA degradation. Avram suggested to test the RNase effect also on lysozyme, a protein that was used as our second model substrate. Here we got no effect, which was kind of a surprise, as proteolysis of the two substrates, BSA and lysozyme, behaved in an identical manner all along the way: ATP as well as all the different factors we resolved from the crude extract, were all required for the degradation of both proteins. Avram suspected that the RNase effect could be an artifact. Meanwhile APF-1 was identified by Keith Wilkinson and his colleagues as ubiquitin (see above), and the amino acid sequence/composition of ubiquitin disclosed the “secret” of the dry weight/protein measurement discrepancy – the molecule has a single tyrosine residue. So we decided not to pursue this subject, and the selective inhibitory effect of RNase A on BSA degradation had remained an unsolved mystery – for the time being.
I had not stopped suspecting, however, that the findings must represent some true biological phenomenon, and used the opportunity of my independence at Harvey’s laboratory to pull out the late 1970s results from my notebook and start dissecting the RNase effect in a systematic manner. With some advice from Alexander Varshavsky (Alex; M.I.T.), and a lot of help and reagents from Joan Steitz (Yale), Harvey Lodish and Uttam RajBhandary (M.I.T.), I managed to make some progress. I discovered that the degradation of BSA was completely dependent on specific tRNAs, that of Arg and His, and that the destruction of the tRNA led to inhibition of the reaction. The nature of the mechanism of action of the tRNAs and the problem of why the degradation of lysozyme was insensitive to RNase had remained a mystery at that time, which was resolved only when I returned to Israel and established my own laboratory (see below).
The other ubiquitin subject I was studying involved a collaboration with Alex Varshavsky and his then graduate student, Daniel Finley – Dan. At that time Alex was studying the role of mono-ubiquitination of histones (for the histone-ubiquitin adduct H2A known also as protein A24 or uH2A, see above). Alex noted a series of publications on a temperature sensitive cell cycle arrest mouse mutant cell, ts85, that was generated and described by the group of M. Yamada. These researchers reported that at the non-permissive temperature, the cell lost the histone H2A-ubiquitin adduct. With the ubiquitin cycle unraveled we surmised that this loss could be due to one of two defects: (i) loss of ubiquitination, or (ii) activated de-ubiquitination. Planning our experimental approach, we thought that the defect in these cells is more likely due to loss rather then to gain of function, and we set to dissect the defect. The idea was that the same defect in monoubiquitination of the histone may affect also protein degradation which involves polyubiquitination, though it was clear that the single modification of the histone molecule by ubiquitin does not lead to its targeting to proteolysis. Identification of the biochemical defect in the cells was not too difficult, as we used the isolation technique of the conjugating enzymes developed in Haifa and demonstrated that the defect results from a temperature-sensitive ubiquitin-activating enzyme, E1, the first enzyme in the ubiquitin system cascade (see above). Importantly, inactivation of the enzyme led to inhibition of ubiquitin conjugation to the general population of cellular proteins and was not confined to inhibition of conjugation of histone H2A. Consequently, degradation of both abnormal and normal shortlived proteins was also inhibited, demonstrating that the same enzyme that is involved in ubiquitin activation for histone modification, is also involved in activation of ubiquitin for modification of substrates destined for degradation. We were very lucky in the sense, as if the defect would have been more specific, involving an E3 that targets several substrates, or “worse”, a specific histone E3, we could not have possibly detected an effect on the degradation of the general population of cell proteins: only a defect in a key enzyme such as E1 could have resulted in such a dramatic effect. Identification and characterization of the cell defect further corroborated our earlier general hypothesis that ubiquitination signals proteins for degradation, and that it also occurs in nucleated cells, a finding we had already demonstrated in Haifa, albeit indirectly (using anti-bodies raised against ubiquitin and monitoring the level and dynamics of ubiquitin-protein adducts under conditions of basal and accelerated proteolysis in hepotoma cells; see above). Since the ts85 cell was also a cell cycle arrest mutant, we hypothesized, but did not show at the time experimentally, that the system may be involved in regulating the cell cycle, an hypothesis that later turned out to be correct.