Endoplasmic reticulum I have a very sporadic and idiosyncratic series in which I talk about “classic papers”, and in my idiosyncratic series Vic Engelhard’s paper on tyrosinase processing counts as a classic paper. It was one of the early indications that proteins in the ER must be degraded in the cytosol, and as such it’s one of a number of ways that antigen presentation has helped fundamental understanding of cell biology; but I think it hasn’t received as much recognition as it could have.

But perhaps I should begin at the beginning.

Proteolysis is a normal part of cell function. Proteins that are damaged, misfolded, or mis-translated, as well as proteins that have simply reached the end of their useful life, are degraded and converted to amino acids that can be recycled into new proteins. In the early- to mid-1990s, there were three general systems that were known to degrade proteins, depending on which subcellular compartment they were in:

  • In the cytosol and nucleus, proteins are predominately degraded by proteasomes.
  • Proteins taken up from the exterior of the cell can be degraded in acidic lysosomes
  • Mitochondrial proteins are degraded by a number of proteases within the mitochondrion1

Endoplasmic reticulum (pancreas cell)That leaves an obvious gap. What happens to proteins that are in the endoplasmic reticulum (ER)? This is particularly relevant because the ER is a ferociously active site of protein synthesis, folding, and assembly; when any of those steps goes awry, the protein is supposed to be degraded, a process known as “quality control”. It was clear in the 1980s that proteins that failed quality control in the ER were degraded; in human cells, a well-known example was the cystic fibrosis transmembrane conductance regulator (CFTR), which folds inefficiently and is rapidly degraded2. But it was not clear where the degradation happened (in the ER? The cytosol? Somewhere else?), and what proteases were responsible.

At first it was believed that “what happens in the ER stays in the ER” — ER proteins were degraded in the ER, by ill-defined proteases in that compartment. But I don’t think there was much enthusiasm for that belief, and basically, the field was a mess, as you can see from this introductory paragraph from the time:3

Other membrane proteins are also known to be degraded at the ER, but the process is poorly understood, and the responsible enzymes have not been identified. For example, some of these proteolytic events are ATP dependent, but some are not; some occur within the lumen while others take place on the cytoplasmic side; some exhibit inhibitor sensitivities characteristic of serine proteases, whereas others do not.

This was relevant to me as an immunologist, because viral glycoproteins (which, of course, are synthesized in the ER) are popular targets for cytotoxic T lymphocyte (CTL) recognition (a quick review of MHC class I antigen presentation is here). We knew in the early 1990s that most if not all CTL targets — even those derived from ER proteins — were generated in the cytosol. As I wrote in a 1996 review:4

Proteins targeted into the ER by signal sequences can also be presented on MHC I molecules. Since these molecules are cotranslationally transported into the endoplasmic reticulum, they might be expected to bypass hydrolysis in the cytosol. However, where analyzed, the presentation of most of these antigens is dependent on the TAP-transporter and on proteasome activity, and therefore the presented peptides are probably being generated in the cytosol.

The three obvious possible explanations were that either the putative glycoprotein never made it into the ER and was degraded as a mistargeted protein (Jon Yewdell would call that a “DRiP”; I can’t remember exactly when I heard him propose that first, but it was around that time); that the glycoprotein went in to the ER, got degraded there, and the peptides were first transfered to the cytosol; or that the glycoprotein was transfered from the ER to the cytosol and degraded there.

The simplest explanation, at the time, seemed to be the first one –  proteins never made it in to the ER, and were degraded in the cytosol. However, it wasn’t an explanation that we liked very much, for various reasons. Vic Engelhard’s paper (Remember Engelhard’s paper? This here’s a post about Engelhard’s paper) cleared that question up, at least for one epitope.

Skipper, J. C., Hendrickson, R. C., Gulden, P. H., Brichard, V., Van Pel, A., Chen, Y., Shabanowitz, J., Wolfel, T., Slingluff, C. L., Jr., Boon, T., Hunt, D. F., and Engelhard, V. H. (1996). An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins. J. Exp. Med. 183, 527-534.

His finding is simple enough to describe, although it relied on a technically very difficult mass spec analysis:

  1. A peptide presented on MHC class I was derived from an ER protein (which they knew from its sequence);
  2. the protein had actually gone into the ER, because it had been N-glycosylated, which only happens in the ER;
  3. yet the peptide itself was probably generated in the cytosol, because enzymes that modified it are mainly found in the cytosol.

Mumps virus protein (turquoise) in endoplasmic reticulumThe most surprising and exciting part was the second point: Clear evidence that the protein had actually gone into the ER before the peptide was generated. 5 This wasn’t by any means definitive proof that ER proteins are degraded in the cytosol (a follow-up paper in 19986 took it a bit further) but it certainly was suggestive.

If Vic’s paper had come out a year or two earlier, it would probably have made much more of a splash than it did, but in February of 1996 it was only a nose ahead of several more focused papers. The field had started to clear up in the mid-1990s, with some observations in yeast in 1993 and 1994 7 and around 1995 moving into mammalian cells with (among others) the Jensen et al. paper I quoted above. 8  And later in 1996, the iceberg tipped over altogether, with a whole bunch of almost simultaneous papers that showed quite clearly that ER degradation wasn’t done by ER proteases at all, but was in fact performed by proteasomes. 9 All the papers demonstrated that there’s an export step before degradation: ER proteins that fail quality control are shunted out into the cytosol, where the proteasomes can grab onto them and chop them up. (The export step is still not all that well understood in molecular detail, though in 2007 it started to open up some, I think.)

Though Engelhard’s 1996 paper is reasonably widely cited (256 citations as I write this) it clearly didn’t have the impact on cell biology in general that it did on me, probably because it came out around the same time as a bunch of more specific papers. This blogpost is an attempt to give a bit more retroactive credit to a very nice example of logical reasoning from indirect evidence.

  1. I believe that some, though not all, of the mitochondrial proteases were identified in the late 1980s/early 1990s, and that the broad outline of mitochondrial proteolysis was understood in the early 1980s (Desautels, M. and Goldberg, A. L. (1982) Liver mitochondria contain an ATP-dependent, vanadate-sensitive pathway for the degradation of proteins. Proc Natl Acad Sci USA 79 , pp. 1869-1873.). I don’t know all that much about mitochondrial proteolysis, though, so if someone wants to correct me, please do so.[]
  2. For example, Ward C, Kopito R (October 14, 1994) Intracellular turnover of cystic fibrosis transmembrane conductance regulator. Inefficient processing and rapid degradation of wild-type and mutant proteins. J. Biol. Chem. 269.:25710-25718[]
  3. Taken from Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, et al. (October 6, 1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83:129-35. with references removed[]
  4. York, I. A., and Rock, K. L. (1996). Antigen processing and presentation by the class I major histocompatibility complex. Annual review of immunology Annu Rev Immunol 14, 369-396.[]
  5. Technical explanation: The mass spec analysis showed that the asparagine that is encoded in the DNA was actually an aspartic acid in the presented peptide; deglycosylating enzymes that were believed to only be present in the cytosol remove carbohydrates from Asn to generate Asp.[]
  6. Mosse, C. A., Meadows, L., Luckey, C. J., Kittlesen, D. J., Huczko, E. L., Slingluff, C. L., Shabanowitz, J., Hunt, D. F., and Engelhard, V. H. (1998). The class I antigen-processing pathway for the membrane protein tyrosinase involves translation in the endoplasmic reticulum and processing in the cytosol. J Exp Med 187, 37-48.[]
  7. (Sommer T, Jentsch S (September 9, 1993) A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum. Nature 365.:176-9.
    Kölling R, Hollenberg CP (July 15, 1994) The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants. EMBO J 13.:3261-71.[]
  8. Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, et al. (October 6, 1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83:129-35. []
  9. I may be missing some:
    Hampton RY, Gardner RG, Rine J (December 1996) Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein. Mol Biol Cell 7.:2029-44.
    Werner ED, Brodsky JL, McCracken AA (November 26, 1996) Proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate. Proc Natl Acad Sci U S A 93.:13797-801.
    Hiller MM, Finger A, Schweiger M, Wolf DH (September 20, 1996) ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway. Science 273.:1725-8.
    Qu D, Teckman JH, Omura S, Perlmutter DH (September 13, 1996) Degradation of a mutant secretory protein, alpha1-antitrypsin Z, in the endoplasmic reticulum requires proteasome activity. J Biol Chem 271.:22791-5.
    Wiertz EJ, Jones TR, Sun L, Bogyo M, Geuze HJ, et al. (March 8, 1996) The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell 84.:769-79.[]