Yeast as a model to understand mechanisms and consequences of protein modifications by metabolism derived toxic products.

An inevitable consequence of metabolism in cells across all kingdoms of life is the non-enzymatic formation of toxic metabolites such as reactive oxygen species (ROS) and reducing sugars, whose reaction products are associated with several diseases such as clinical complications of diabetes, cataracts, cancer and age-related late-onset diseases like Parkinson's Disease and Alzheimer's Disease, amongst others. Yeast has been used successfully to study the reaction of these toxic metabolites with proteins in vivo, which I will summarize with exemplified studies on 2 crucial reactions, protein carbonylation and protein glycation.

Assembly of heterodimeric luciferase after de novo synthesis of subunits in rabbit reticulocyte lysate involves hsc70 and hsp40 at a post-translational stage.

Heterodimeric luciferase from Vibrio harveyi had been established as a unique model enzyme for direct measurements of the effects of molecular chaperones and folding catalysts on protein folding and subunit assembly after de novo synthesis of subunits in rabbit reticulocyte lysate. It was observed that luciferase assembly can be separated in time from synthesis of the two subunits and that under these post-translational conditions assembly was inhibited by either ATP depletion or inhibition of peptidylprolyl cis/trans isomerases, that is, by addition of cyclosporin A or FK506. Furthermore, it was observed that the inhibitory effect of FK506 on luciferase assembly can be suppressed by addition of purified cyclophilin, thereby providing the first direct evidence for the involvement of peptidylprolyl cis/trans isomerases in protein biogenesis in the eukaryotic cytosol. Here the ATP requirement in luciferase assembly has been characterized. Depletion of either Hsp90 or CCT from reticulocyte lysate did not interfere with luciferase assembly. However, addition of purified Hsc70 stimulated luciferase assembly. While addition of purified Hsp40 did not have any effect on luciferase assembly, the stimulatory effect of Hsc70 was further increased by Hsp40. Thus, after synthesis of the two subunits in reticulocyte lysate assembly of heterodimeric luciferase involves Hsc70 and its co-chaperone Hsp40. Therefore, Hsc70 aids protein biogenesis in the eukaryotic cytosol not only at the levels of nascent polypeptide chains and precursor proteins that have to be kept competent for transport into cell organelles, but also at the level of subunits that have to be kept competent for assembly.

Homologs of the yeast Sec complex subunits Sec62p and Sec63p are abundant proteins in dog pancreas microsomes.

Cotranslational protein transport into dog pancreas microsomes involves the Sec61p complex plus a luminal heat shock protein 70. Posttranslational protein transport into the yeast endoplasmic reticulum (ER) involves the so-called Sec complex in the membrane, comprising a similar Sec61p subcomplex, the putative signal peptide receptor subcomplex, and the heat shock protein 40-type subunit, Sec63p, plus a luminal heat shock protein 70. Recently, human homologs of yeast proteins Sec62p and Sec63p were discovered. Here we determined the concentrations of these two membrane proteins in dog pancreas microsomes and observed that the canine homologs of yeast proteins Sec62p and Sec63p are abundant proteins, present in almost equimolar concentrations as compared with Sec61alphap monomers. Furthermore, we detected fractions of these two proteins in association with each other as well as with the Sec61p complex. The J domain of the human Sec63p was shown to interact with immunoglobulin heavy chain binding protein. Thus, the membrane of the mammalian ER contains components, known from the posttranslationally operating protein translocase in yeast. We suggest that these components are required for efficient cotranslational protein transport into the mammalian ER as well as for other transport processes.

Efficient folding of firefly luciferase after transport into mammalian microsomes in the absence of luminal chaperones and folding catalysts.

Folding of polypeptides emerging from the protein translocase in the membrane of mammalian microsomes was analyzed after synthesis of corresponding precursor proteins in a mammalian translation system. Firefly luciferase was used as a model protein; the corresponding hybrid precursor contained the preprolactin signal peptide. The rates and efficiencies of folding of luciferase in microsomes were compared with those of folding of luciferase in the cytosol. Furthermore, folding of luciferase in microsomes was compared with that in proteoliposomes, i.e. in the absence of luminal molecular chaperones and folding catalysts. Folding in microsomes was less efficient compared with folding in the cytosol. Folding in the absence of luminal proteins was more efficient compared with folding in their presence and identical to folding in the cytosol. Thus, firefly luciferase emerging from translocase can efficiently fold to its native conformation without chaperoning by any luminal proteins. There may be molecular chaperones present in the microsomal membrane that can efficiently substitute for the cytosolic chaperone machinery comprising Hsp40, Hsp60, and Hsp70 with respect to folding of firefly luciferase.

Protein folding within and protein transport into mammalian microsomes are differentially affected by photoaffinity labeling of microsomes with 8-azido-ATP.

Transport of presecretory proteins into mammalian microsomes involves a microsomal protein which is sensitive to photoaffinity labeling with 8-azido-ATP. Typically, protein folding within the lumen of the endoplasmic reticulum of mammalian cells depends on ATP and the member of the Hsp70 protein family, BiP. Here we addressed the question of whether protein transport into and folding within microsomes are differentially affected by photoaffinity labeling of microsomes with 8-azido-ATP. Folding of heterodimeric luciferase to the native state was more azido-ATP-sensitive compared to transport of the precursors of the two subunits. Therefore, we conclude that the microsomal protein which is responsible for the ATP-dependence of protein folding in the endoplasmic reticulum is sensitive to photoaffinity labeling with 8-azido-ATP and that this microsomal protein is distinct from the microsomal ATP-binding protein which is involved in protein transport.