Substrate-specific regulation of the ribosome– translocon junction by N-terminal signal sequences

Amino-terminal signal sequences target nascent secretory and membrane proteins to the endoplasmic reticulum for translocation. Subsequent interactions between the signal sequence and components of the translocation machinery at the endoplasmic reticulum are thought to be important for the productive engagement of the translocon by the ribosome-nascent chain complex. However, it is not clear whether all signal sequences carry out these posttargeting steps identically, or if there are differences in the interactions directed by one signal sequence versus another. In this study, we find substantial differences in the ability of signal sequences from different substrates to mediate closure of the ribosome–translocon junction early in translocation. We also show that these differences in some cases necessitate functional coordination between the signal sequence and mature domain for faithful translocation. Accordingly, the translocation of some proteins is sensitive to replacement of their signal sequences. In a particularly dramatic example, the topology of the prion protein was found to depend highly on the choice of signal sequence used to direct its translocation. Taken together, our results reveal an unanticipated degree of substrate-specific functionality encoded in N-terminal signal sequences.

Heat-shock protein 104 expression is sufficient for thermotolerance in yeast.

In all organisms, mild heat pretreatments induce tolerance to high temperatures. In the yeast Saccharomyces cerevisiae, such pretreatments strongly induce heat-shock protein (Hsp) 104, and hsp104 mutations greatly reduce high-temperature survival, indicating Hsp1O4 plays a critical role in induced thermotolerance. Surprisingly, however, a heat-shock transcription factor mutation (hsf1-m3) that blocks the induction of Hsps does not block induced thermotolerance. To resolve these apparent contradictions, we reexamined Hsp expression in hsf1-m3 cells. HsplO4 was expressed at a higher basal level in this strain than in other S. cerevisiae strains. Moreover, whereas the hsf1-m3 mutation completely blocked the induction of Hsp26 by heat, it did not block the induction of Hsp1O4. HSP104 could not be deleted in hsf1-m3 cells because the expression of heat-shock factor (and the viability of the strain) requires nonsense suppression mediated by the yeast prion [PSI+], which in turn depends upon Hsp1O4. To determine whether the level of Hsp1O4 expressed in hsf1-m3 cells is sufficient for thermotolerance, we used heterologous promoters to regulate Hsp1O4 expression in other strains. In the presence of other inducible factors (with a conditioning pretreatment), low levels of Hsp1O4 are sufficient to provide full thermotolerance. More remarkably, in the absence of other inducible factors (without a pretreatment), high levels of Hsp1O4 are sufficient. We conclude that Hsp1O4 plays a central role in ameliorating heat toxicity. Because Hsp1O4 is nontoxic and highly conserved, manipulating the expression of Hsp1OO proteins provides an excellent prospect for manipulating thermotolerance in other species.

Viral particles are required for infection in neurodegenerative Creutzfeldt-Jakob disease.

Several models have been proposed for the infectious agents that cause human Creutzfeldt-Jakob disease (CJD) and sheep scrapie. Purified proteins and extracted nucleic acids are not infectious. To further identify the critical molecular components of the CJD agent, 120S infectious material with reduced prion protein (PrP) was treated with guanidine hydrochloride or SDS. Particulate and soluble components were then separated by centrifugation and molecularly characterized. Conditions that optimally solubilized residual PrP and/or nucleic acid-protein complexes were used to produce subfractions that were assayed for infectivity. All controls retained > 90% of the 120S titer (approximately 15% of that in total brain) but lost > 99.5% of their infectivity after heat-SDS treatment (unlike scrapie fractions enriched for PrP). Exposure to 1% SDS at 22 degrees C produced particulate nucleic acid-protein complexes that were almost devoid of host PrP. These sedimenting complexes were as infectious as the controls. In contrast, when such complexes were solubilized with 2.5 M guanidine hydrochloride, the infectious titer was reduced by > 99.5%. Sedimenting PrP aggregates with little nucleic acid and no detectable nucleic acid-binding proteins had negligible infectivity, as did soluble but multimeric forms of PrP. These data strongly implicate a classical viral structure, possibly with no intrinsic PrP, as the CJD infectious agent. CJD-specific protective nucleic acid-binding protein(s) have already been identified in 120S preparations, and preliminary subtraction studies have revealed several CJD-specific nucleic acids. Such viral candidates deserve more attention, as they may be of use in preventing iatrogenic CJD and in solving a fundamental mystery.