Prions Other molecules besides organelle DNA are inherited in non Mendelian patterns Examples of non Mendelian patterns of inheritance extend beyond the inheritance of organelle DNA Certain DNA and RNA plasmids for example are inherited in non Mendelian patterns but the strangest cases of non Mendelian inheritance is the inheritance of endogenous proteins with altered structures these proteins are known as prions Prions can be inherited in yeast and the filamentous fungus Podospera anserina and most of our discussion will focus on two elements in yeast known as URE3 and PSI Prions were first proposed to cause spongiform encephalopathies in mammals Prions were first discovered as the etiological agents in transmissible spongiform encephalopathies such as scrapie in sheep and goats bovine spongiform encephalopathy BSE or mad cow disease in cattle and Creutzfeldt Jacob disease CJD Gerstmann Straussler Schneinker syndrome GSS fatal familial insomnia and kuru in humans Collectively these diseases have been explained by the prion hypothesis This hypothesis posits that the infectious agent is an abnormal form of a cellular protein known as PrP During the course of the prion disease a protease resistant aggregated form of PrP designated PrPSc accumulates in the brain The model proposes that the presence of PrPSc which has the same sequence as PrP but a different structure will recruit normal PrP into the abnormal structure to spread the disease The disease is thought to spread when animals are fed food containing brain material Alternatively with rare genetic forms of the disease individuals inherit a mutant form of PrP that has a higher probability of spontaneously folding into a protease resistant prion form In 1997 Stanley Prusiner a neurologist at UCSF won the Nobel Prize in Physiology and Medicine for his work leading to the prion hypothesis Below are several results for this class of diseases that are consistent with the prion hypothesis 1 No nucleic acid has ever been shown to de associated with the purified Scrapie infectious agent Purified PrPSc can cause Scrapie in many animals including mice 2 Familial spongiform encephalopathies in humans are associated with mutations in the PrP gene About 10 of human prion diseases are hereditary and these are associated with mutations in the PrP gene Individuals affected by GSS for example contain a Pro to Leu mutation in their PrP gene Transgenic mice that carry a mouse PrP gene with the GSS lesion will spontaneously develop spongiform disease Normal mice carrying the wild type PrP gene do not 3 Mice lacking the PrP gene are normal but are resistant to Scrapie All of these results are consistent with the prion hypothesis Further evidence for the prion hypothesis Evidence of a different type supporting the prion hypothesis has come from experiments in fungi The yeast element URE3 for example can now be explained by the prion hypothesis In yeast mutations in the nuclear gene for aspartate transcarbamylase an enzyme in the pyrimidine biosynthetic pathway that produces ureidosuccinate from carbamyl phosphate and aspartate cause an auxotrophic phenotype that can be rescued by growing on exogenous ureidosuccinate in low nitrogen Yeast growing on a rich nitrogen source such as ammonia repress transcription of enzymes and transporters needed for the utilization of poor nitrogen sources The Ure2 protein senses the presence of a rich nitrogen source and blocks the action of the Gln3 protein a positive transcriptional regulator of many genes whose products facilitate the utilization of poor nitrogen sources One of the genes that Gln3p regulates is DAL5 which encodes a protein that can import ureidosuccinate into the cell Thus cells mutant for aspartate transcarbamylase will not grow on exogenous ureidosuccinate in the presence of ammonia because Gln3 is inactivated by Ure2 protein In the absence of active Gln3 the Dal5 transporter is not produced Aspartate transcarbamylase mutant grown on exogenous ureidosuccinate URE2 genotype nitrogen growth NH3 NH3 ure2 NH3 URE3 NH3 In the 1970s Lacroute identified mutations in the gene URE2 on the basis of this phenotype mutants that would now grow in the absence of the aspartate transcarbamylase gene and the presence of ammonia and ureidosuccinate ure2 mutations segregate 2 2 as a Mendelian trait Other mutants with the same phenotype called URE3 were also isolated but these elements are not inherited in a Mendelian pattern For example crosses between wild type and URE3 mutants generated diploids that were URE3 Hence the mutant appeared dominant Sporulation of the URE3 diploids however usually generated 4 URE3 0 wild type spores although other inheritance patterns were also seen This inheritance pattern of URA3 is clearly non Mendelian These experiments suggested that URE3 was mitochondrial but when mitochondrial DNA was lost in these strains URE3 was still present Lacroute carried out these experiments in the early 1970s and didn t have a good explanation for URE3 In the 1990s Reed Wickner tested the idea that URE3 was a prion form of the Ure2 protein Several experiments supported the prion hypothesis First increasing the amount of Ure2 protein by 10 fold increases the frequency of spontaneous URE3 mutants generated by 100 fold Second yeast cells containing URE3 can be cured of this element by growth in low concentrations of guanidine a protein denaturant Third the normal Ure2 protein is susceptible to protease but resistant in a URE3 background Fourth the URE2 gene and thus the Ure2 protein was also shown to be required for the activity of URE3 URE3 is lost when placed in a ure2 mutant background Additional evidence for the prion hypothesis comes from PSI which is likely to be a prion form of the Sup35 protein a translational terminator Like URE3 PSI is a non Mendelian element that requires the presence of the SUP35 gene for its effects The PSI phenotype occurs at a much higher frequency in yeast that overexpress Sup35 protein similar to the relationship between URE3 and the Ure2 protein Removal of the gene encoding the chaperone protein Hsp104 cures yeast of PSI The involvement of a protein folding chaperone in the maintenance of PSI argues strongly that PSI is a protein structure These genetic a biochemical results are all consistent with URE3 and PSI being prion forms of the Ure2 and Sup35 proteins It is interesting that the portions of the Ure2 and Sup35 proteins involved in prion formation are distinct
View Full Document
Unlocking...