In a new and dynamic field, everything-you-know-is-wrong papers appear regularly, and no one is too surprised. Usually, once a field of study has been around for a while (twenty years or more, say) most of the basics are settled in, and when an e-y-k-i-w paper comes along there’s either great skepticism or great angst or both. But there are also some long-established fields where paradigms seem to be shattered on a weekly basis. Ubiquitin is one of those. Another universal rule of ubiquitin was disproven recently, and I for one just nodded thoughtfully, unsurprised.

The paper is:

Ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce ERAD of MHC-I by viral E3 ligase mK3

Xiaoli Wang, Roger A. Herr, Wei-Jen Chua, Lonnie Lybarger, Emmanuel J.H.J. Wiertz, and Ted H. Hansen

The Journal of Cell Biology, Vol. 177, No. 4, May 21, 2007 613-6241

It’s particularly interesting to me because it’s yet another example of the important insights into cell biology that arise from antigen presentation in general and viral immune evasion in particular.

The paradigm that was overthrown is that “poly-ubiquitination, the process in which a chain of at least four ubiquitin peptides are attached to a lysine on a substrate protein, most commonly results in the degradation of the substrate protein via the proteasome.” (That’s from Wikipedia– my first source for oversimplified summaries that miss important advances and misinterpret what they do find. But other articles on ubiquitin include similar statements.) It’s the “lysine” bit I’m taking issue with in this case.

Ubiquitin moleculeUbiquitin/proteasome pathwayUbiquitin was identified 30-odd years ago (picture on the left from the Nobel Prize web site). It’s a small, abundant protein that’s found in all eukaryotes, and it’s involved in protein destruction. That’s the last of the firm statements: For the rest of this paragraph, you should imagine every statement to be footnoted or qualified in some way, because throughout the past 30 years ubiquitin has made a habit of constantly revealing unexpected functions and new aspects. The simplest pattern is the one you’ll find in innumerable posters and illustrations (the one on the right is from Sigma-Aldrich, but there are scores of virtually-identical ones out there). In this pathway, ubiquitin is covalently attached to proteins, new ubiquitins are attached to the original one, a polyubiquitin chain forms, the proteasome recognizes the polyubiquitin chain, and the tagged protein is destroyed, releasing ubiquitin to kill again. It’s one way to put the regulation in your regulated proteolysis.

Poly-ubiquitin chain on Src

The canonical linkage for ubiquitin in this targeting to the proteasome is between a lysine on a substrate protein, and the terminal glycine on ubiquitin; followed by a chain of ubiquitins tagging onto the preceding ubiquitin’s lysine 48. The beautiful picture on the right is taken from the PDB’s Molecule of the Month from 2004, and shows “a string of ubiquitin molecules (colored pink and tan here, from PDB entry 1ubq) attached to old proteins, such as the src protein shown here (colored blue, from PDB entry 2src).”

There’s no room here to talk about all of the myriad other functions for ubiquitin that have been discovered over the years, but I want to highlight one in particular. In the mid 1990s2 it was discovered that ubiquitination of cell-surface molecules didn’t necessarily lead to destruction by the proteasome, but rather to internalization and in some cases destruction by the lysosome. What’s more, this receptor targeting mode of ubiquitin often involves polyubiquitin chains extending from ubiquitin’s lysine 63, not 48.3 So already there was precedent for flexibility in ubiquitin linkages.

A more recent observation came in the late 1990s and early 2000s, with the unexpected discovery4 that ubiquitin doesn’t even need lysines on its substrate protein; instead, ubiquitin can link up with the amino terminal residue of the protein and form a polyubiquitin chain there.

(I’m surprised that this doesn’t seem to be more widely known. I’ve talked to several people who have come to me, scratching their heads, because they’ve mutated all the lysines in their protein and still see it being ubiquitinated and destroyed — they were quite amazed when I pointed this phenomenon out to them.)

Wang et al, in the paper I’m highlighting here, take this one step further. They were looking at the way mK3 (a viral immune evasion molecule that causes class I major histocompatibility complexes to be rapidly degraded by the proteasome) causes degradation of MHC class I molecules. To make a long story short — hey, you should read the paper yourself! — they mutated all the lysines in the substrate protein and still saw polyubiquitination and degradation. But when they removed threonines and series — amino acids that are supposedly inaccessible to ubiquitin tagging — then the protein was no longer polyubiquitinated, and was no longer degraded. This seems to be a novel chemical process for ubiquitin, too, not involving the usual amide linkage but instead involving an ester bond.

What are the implications of this? On a purely technical basis, of course, it means that all the people who have decided ubiquitin can’t be important for their protein because there are no lysines available, have to go back and actually test directly. A more interesting question is whether non-lysine ubiquitination is a normal cellular process that the virus is just piggy-backing on, or whether this doesn’t occur normally and mK3 somehow forces the system in a new and bizarre direction. My guess is that this is in fact a normal cellular capability (there are hints in the paper and from previous literature that this may not be an abnormal event, but as yet they’re only hints). If so, the next question is whether this is a normal function that’s specific for the particular form of degradation here — that is, ER-associated degradation (ERAD), which is the process by which secreted or transmembrane proteins get destroyed during their maturation in the endoplasmic reticulum. ERAD is a fairly new and active field, and there’s a lot that’s not understood about it yet. If non-lysine ubiquitination is ERAD-specific, or especially if it’s actually a marker for ERAD, that would be really interesting and might offer a handle manipulating ERAD. Wang et al conclude:

It will be interesting to determine whether other ERAD pathways involving transmembrane protein substrates might also involve tail ubiquitination using non-K residues. Furthermore, the fact that mK3 has numerous viral (including MIR1) and cellular homologues makes it attractive to speculate that other ubiquitination-regulated processes use similar nonconventional methods of Ub conjugation.

  1. doi:10.1083/jcb.200611063[]
  2. I think the first papers were Hicke L and Riezman H (1996) Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell, 84, 277-287. and Strous GJ, Vankerkhof P, Govers R, Ciechanover A and Schwartz AL (1996) The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor. EMBO J, 15, 3806-3812.[]
  3. Nice if now dated review: Dubiel W, Gordon C. Ubiquitin pathway: another link in the polyubiquitin chain? Curr Biol. 1999 Jul 29-Aug 12;9(15):R554-7.[]
  4. The EMBO Journal (1998) 17, 5964-5973. A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein. Kristin Breitschopf, Eyal Bengal, Tamar Ziv, Arie Admon and Aaron Ciechanover. (doi:10.1093/emboj/17.20.5964); also see Ciechanover’s review in Trends Cell Biol. 2004 Mar;14(3):103-6 (doi:10.1016/j.tcb.2004.01.004) []