Reed B. Wickner, M.D. : NIDDK

Reed B. Wickner, M.D.

NIDDK, National Institutes of Health
Building 8, Room 207
8 Center Dr.
Bethesda, MD 20892-0830
Tel: 301-496-3452
Fax: 301-402-0240

Research Website:

Education / Previous Training and Experience:
B.A., Cornell University, 1962
M.D., Georgetown University, 1966

Research Statement:

Our studies of infectious elements of S. cerevisiae have led to the discovery of prions, dsRNA viruses, and naked ssRNA replicons, their similarities with similar elements in animal cells and some clues of the mechanisms by which they are propagated and interact with their host.

Prions of yeast

We discovered two prions of the yeast, Saccharomyces cerevisiae. Genetic evidence led us to propose that the non-Mendelian genetic elements, [URE3] and [PSI], are each prions (infectious proteins). A prion is an altered form of a normal protein that may have lost its normal function, but has acquired the ability to convert the normal form of that protein to the abnormal (prion) form. [URE3] is an altered form of the URE2 protein whose normal function is to turn off utilization of poor nitrogen sources if a good nitrogen source is present. [PSI] is the prion form of the SUP35 protein which is involved in translation termination. We have now obtained several lines of evidence that [URE3] and [PSI] are prions. We have found that the prion domain of Ure2p is the N-terminal 65 amino acid residues, a region rich in asparagine, serine and threonine.

We have purified the normal form of Ure2p and find it is a soluble dimer. The Ure2p prion domain (residues 1-65) is unstructured in native Ure2p, but readily forms amyloid in vitro, and, when mixed with the native full length Ure2p, induces the latter to also form amyloid. We have recently shown that amyloid of recombinant Ure2p is infectioius for yeast cells, infecting them with the [URE3] prion. The prion domain is sufficient for infectivity. Cells infected with amyloid of recombinant Ure2p carry any of at least three prion variants. Extracts of a variant are again infectious for yeast and transmit only the variant with which they are infected. Infectious particles are larger than 40 mers.

We find that the amyloid of Ure2p consists of a beta-sheet core region (Ure2 residues 1-65), a C-terminal domain similar in structure to glutathione-S-transferases (Ure2 residues 95-354) and a flexible tether connecting these two regions (Ure2 residues 66-94). In Ure2 amyloid, monomers are connected by interactions between the N-terminal domains. Prion formation in vivo and amyloid formation in vitro are relatively independent of Ure2p prion domain sequence but strongly depend on amino acid content. We recently showed that randomly shuffling the prion domain of Ure2p or of Sup35p and integrating the shuffled prion domain into the chromosome in place of the wild-type sequence produces proteins all of which can still be prions! This means that amino acid content (and not sequence) are the prime determinants of prion forming ability. It also argues that the prion domain assumes a parallel in-register beta sheet structure, not anti-parallel beta sheet or beta helix. Efforts are now underway to confirm this prediction.

We showed that propagation of the [URE3] prion requires the Hsp104 disaggregating chaperone, as well as the Hsp70 family chaperone Ssa2. Overproduction of the Hsp40 chaperone Ydj1p cures [URE3]. We have shown that both [URE3] and [PSI] are diseases of yeast, that in spite of their infectious nature and ability to arise de novo rather often they are not found in a survey of wild strains.

We recently described a new kind of prion, an enzyme whose active form is necessary for the activation of its own inactive precursor. This prion is simply the vacuolar protease B of yeast. It is not novel to find a self-activating protease, but to find a protease acting as a gene is unique. Moreover, many self-modifying proteins are known and some may prove to be this type of prion as well. Recent evidence from Silar and coworkers suggests that a protein kinase in Podospora anserina can be such a prion.

Current areas of concentration include: > further studies of the structure of Ure2p amyloid > efforts to generalize the primacy of composition in prion formation > development of general methods to search for new prions > genetic screens to find proteins affecting prion generation and propagation > genetic screens to find proteins interacting with Ure2p

Double-stranded RNA viruses of yeast

We have developed the classical genetics, molecular cloning and enzymology of the replication of the L-A virus, its satellites and their interactions with the host. We have established template-dependent in vitro replication and transcription systems for this virus, the first for a dsRNA virus, and have used them to elucidate the sites on the RNA necessary and the viral proteins that participate in these processes. We have defined the packaging site on the RNA, the packaging domain on the viral Pol protein and obtained evidence for the mechanism of the packaging process.

The L-A virus has two open reading frames, the 5' gag, encoding the major coat protein, called Gag, and the 3' pol, encoding a multifunctional protein domain called Pol that includes the RNA-dependent RNA polymerase. Pol is expressed only as a Gag-Pol fusion protein formed by a -1 ribosomal frameshift event whose mechanism is identical to that used by retroviruses for the same purpose. We have described chromosomal genes whose mutation alters the efficiency of ribosomal frameshifting and thereby impairs virus propagation. We discovered a set of chromosomal genes, SKI1, SKI2, SKI3, SKI6, SKI7 and SKI8, which repress viral propagation and prevent the L-A virus and its satellite RNAs from harming the cell. Our data indicate that SKI2, SKI3, SKI6, SKI7 and SKI8 control viral propagation by inhibiting the translation of the viral messages because they lack 3'poly(A).

Selected Publications:

Pierce MM, Baxa U, Steven AC, Bax A, Wickner RB Is the prion domain of soluble Ure2p unstructured? Biochemistry (44): 321-8, 2005. [Full Text/Abstract]

Ross ED, Edskes HK, Terry MJ, Wickner RB Primary sequence independence for prion formation. Proc Natl Acad Sci U S A (102): 12825-30, 2005. [Full Text/Abstract]

Ross ED, Minton A, Wickner RB Prion domains: sequences, structures and interactions. Nat Cell Biol (7): 1039-44, 2005. [Full Text/Abstract]

Brachmann A, Baxa U, Wickner RB Prion generation in vitro: amyloid of Ure2p is infectious. EMBO J (24): 3082-92, 2005. [Full Text/Abstract]

Nakayashiki T, Kurtzman CP, Edskes HK, Wickner RB Yeast prions [URE3] and [PSI+] are diseases. Proc Natl Acad Sci U S A (102): 10575-80, 2005. [Full Text/Abstract]

Wickner RB, Edskes HK, Ross ED, Pierce MM, Baxa U, Brachmann A, Shewmaker F Prion genetics: new rules for a new kind of gene. Annu Rev Genet (38): 681-707, 2004. [Full Text/Abstract]

Baxa U, Taylor KL, Steven AC, Wickner RB Prions of Saccharomyces and Podospora. Contrib Microbiol (11): 50-71, 2004. [Full Text/Abstract]

Wickner RB, Edskes HK, Ross ED, Pierce MM, Shewmaker F, Baxa U, Brachmann A Prions of yeast are genes made of protein: amyloids and enzymes. Cold Spring Harb Symp Quant Biol (69): 489-96, 2004. [Full Text/Abstract]

Ross ED, Wickner RB Prions of yeast fail to elicit a transcriptional response. Yeast (21): 963-72, 2004. [Full Text/Abstract]

Wickner RB, Edskes HK, Roberts BT, Baxa U, Pierce MM, Ross ED, Brachmann A Prions: proteins as genes and infectious entities. Genes Dev (18): 470-85, 2004. [Full Text/Abstract]

Wickner RB Edskes HK Roberts BT Baxa U Pierce MM Ross ED Brachmann A Prions: proteins as genes and infectious entities. Genes Dev (18): 470-85, 2004. [Full Text/Abstract]

Ross ED, Baxa U, Wickner RB Scrambled prion domains form prions and amyloid. Mol Cell Biol (24): 7206-13, 2004. [Full Text/Abstract]

Baxa U, Ross PD, Wickner RB, Steven AC The N-terminal prion domain of Ure2p converts from an unfolded to a thermally resistant conformation upon filament formation. J Mol Biol (339): 259-64, 2004. [Full Text/Abstract]

Roberts BT, Moriyama H, Wickner RB URE3] prion propagation is abolished by a mutation of the primary cytosolic Hsp70 of budding yeast. Yeast (21): 107-17, 2004. [Full Text/Abstract]

Baxa U, Taylor KL, Wall JS, Simon MN, Cheng N, Wickner RB, Steven AC Architecture of Ure2p prion filaments: the N-terminal domains form a central core fiber. J Biol Chem (278): 43717-27, 2003. [Full Text/Abstract]

Roberts BT Wickner RB Heritable activity: a prion that propagates by covalent autoactivation. Genes Dev (17): 2083-7, 2003. [Full Text/Abstract]

Edskes HK Wickner RB Conservation of a portion of the S. cerevisiae Ure2p prion domain that interacts with the full-length protein. Proc Natl Acad Sci U S A (99 Suppl 4): 16384-91, 2002. [Full Text/Abstract]

Naitow H Tang J Canady M Wickner RB Johnson JE L-A virus at 3.4 A resolution reveals particle architecture and mRNA decapping mechanism. Nat Struct Biol (9): 725-8, 2002. [Full Text/Abstract]

Baxa U Speransky V Steven AC Wickner RB Mechanism of inactivation on prion conversion of the Saccharomyces cerevisiae Ure2 protein. Proc Natl Acad Sci U S A (99): 5253-60, 2002. [Full Text/Abstract]

Wickner RB Edskes HK Roberts BT Pierce M Baxa U Prions of yeast as epigenetic phenomena: high protein Adv Genet (46): 485-525, 2002. [Full Text/Abstract]

Searfoss A Dever TE Wickner R Linking the 3'' poly(A) tail to the subunit joining step of translation initiation: relations of Pab1p, eukaryotic translation initiation factor 5b (Fun12p), and Ski2p-Slh1p. Mol Cell Biol (21): 4900-8, 2001. [Full Text/Abstract]

Umland TC Taylor KL Rhee S Wickner RB Davies DR The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p. Proc Natl Acad Sci U S A (98): 1459-64, 2001. [Full Text/Abstract]

Searfoss AM Wickner RB 3'' poly(A) is dispensable for translation. Proc Natl Acad Sci U S A (97): 9133-7, 2000. [Full Text/Abstract]

Moriyama H Edskes HK Wickner RB URE3] prion propagation in Saccharomyces cerevisiae: requirement for chaperone Hsp104 and curing by overexpressed chaperone Ydj1p. Mol Cell Biol (20): 8916-22, 2000. [Full Text/Abstract]

Taylor KL, Cheng N, Williams RW, Steven AC, Wickner RB Prion domain initiation of amyloid formation in vitro from native Ure2p. Science (283): 1339-43, 1999. [Full Text/Abstract]

Edskes HK, Gray VT, Wickner RB The [URE3] prion is an aggregated form of Ure2p that can be cured by overexpression of Ure2p fragments. Proc Natl Acad Sci U S A (96): 1498-503, 1999. [Full Text/Abstract]

Masison DC, Maddelein ML, Wickner RB The prion model for [URE3] of yeast: spontaneous generation and requirements for propagation. Proc Natl Acad Sci U S A (94): 12503-8, 1997. [Full Text/Abstract]

Masison DC Blanc A Ribas JC Carroll K Sonenberg N Wickner RB Decoying the cap- mRNA degradation system by a double-stranded RNA virus and poly(A)- mRNA surveillance by a yeast antiviral system. Mol Cell Biol (15): 2763-71, 1995. [Full Text/Abstract]

Masison DC, Wickner RB Prion-inducing domain of yeast Ure2p and protease resistance of Ure2p in prion-containing cells. Science (270): 93-5, 1995. [Full Text/Abstract]

Wickner RB URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae. Science (264): 566-9, 1994. [Full Text/Abstract]

Page last updated: December 18, 2008

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