Ero1α requires oxidizing and normoxic conditions to localize to the mitochondria-associated membrane (MAM)

Protein secretion from the endoplasmic reticulum (ER) requires the enzymatic activity of chaperones and oxidoreductases that fold polypeptides and form disulfide bonds within newly synthesized proteins. The best-characterized ER redox relay depends on the transfer of oxidizing equivalents from molecular oxygen through ER oxidoreductin 1 (Ero1) and protein disulfide isomerase to nascent polypeptides. The formation of disulfide bonds is, however, not the sole function of ER oxidoreductases, which are also important regulators of ER calcium homeostasis. Given the role of human Ero1α in the regulation of the calcium release by inositol 1,4,5-trisphosphate receptors during the onset of apoptosis, we hypothesized that Ero1α may have a redox-sensitive localization to specific domains of the ER. Our results show that within the ER, Ero1α is almost exclusively found on the mitochondria-associated membrane (MAM). The localization of Ero1α on the MAM is dependent on oxidizing conditions within the ER. Chemical reduction of the ER environment, but not ER stress in general leads to release of Ero1α from the MAM. In addition, the correct localization of Ero1α to the MAM also requires normoxic conditions, but not ongoing oxidative phosphorylation.     

Glycoprotein Folding in the Endoplasmic Reticulum

Our understanding of eukaryotic protein folding in the endoplasmic reticulum has increased enormously over the last 5 years. In this review, we summarize some of the major research themes that have captivated researchers in this field during the last years of the 20th century. We follow the path of a typical protein as it emerges from the ribosome and enters the reticular environment. While many of these events are shared between different polypeptide chains, we highlight some of the numerous differences between proteins, between cell types, and between the chaperones utilized by different ER glycopro-teins. Finally, we consider the likely advances in this field as the new century unfolds and we address the prospect of a unified understanding of how protein folding, degradation, and translation are coordinated within a cell.     

Structural and functional link between the mitochondrial network and the endoplasmic reticulum

Mitochondrial and endoplasmic reticulum (ER) networks are fundamental for the maintenance of cellular homeostasis and for determination of cell fate under stress conditions. Recent structural and functional studies revealed the interaction of these networks. These zones of close contact between ER and mitochondria called MAM (mitochondria associated membranes) support communication between the two organelles including bioenergetics and cell survival. The existence of macromolecular complexes in these contact sites has also been revealed. In this contribution, we will review: (i) the ER and mitochondria structure and their dynamics, (ii) the basic principles of ER mitochondrial Ca²⁺ transport, (iii) the physiological/pathological role of this cross-talk.     

5S rRNA-assisted DnaK refolding

Although accumulating evidence has revealed that most proteins can fold without the assistance of molecular chaperones, little attention has been paid to other types of chaperoning macromolecules. A variety of proteins interact with diverse RNA molecules in vivo, suggesting a potential role of RNAs for folding of their interacting proteins. Here we show that the in vitro refolding of a representative molecular chaperone, DnaK, an Escherichia coli homolog of Hsp70, could be assisted by its interacting 5S rRNA. The folding enhancement occurred in RNA concentration and its size dependent manner whereas neither the RNA with the reverse sequence of 5S rRNA nor the RNase pretreated 5S rRNA stimulated the folding in vitro. Based on our results, we propose that 5S rRNA could exert the chaperoning activity on DnaK during the folding process. The results suggest an interesting possibility that the folding of RNA-interacting proteins could be assisted by their cognate RNA ligands.     

Synthetic embryonic lethality upon deletion of the ER cochaperone p58 and the ER stress sensor ATF6α

The unfolded protein response (UPR) is activated as a consequence of alterations to ER homeostasis. It upregulates a group of ER chaperones and cochaperones, as well as other genes that improve protein processing within the secretory pathway. The UPR effector ATF6α augments—but is not essential for—maximal induction of ER chaperones during stress, yet its role, if any, in protecting cellular function during normal development and physiology is unknown. A systematic analysis of multiple tissues from Atf6α−/− mice revealed that all tissues examined were grossly insensitive to loss of ATF6α. However, combined deletion of ATF6α and the ER cochaperone p58^IPK resulted in synthetic embryonic lethality. These findings reveal for the first time that an intact UPR can compensate for the genetic impairment of protein folding in the ER in vivo. The also expose a role for p58^IPK in normal embryonic development.     

Translocons, thermodynamics, and the folding of membrane proteins

Recent three-dimensional structures of helical membrane proteins present new challenges for the prediction of structure from amino acid sequence. Membrane proteins reside stably in a thermodynamic free energy minimum after release into the membrane's bilayer fabric from the translocon complex. This means that structure prediction is primarily a problem of physical chemistry. But the folding processes within the translocon must also be considered. A distilled overview of the physical principles of membrane protein stability is presented, and extended to encompass translocon-assisted folding.     

Interaction of trigger factor with the ribosome

The trigger factor of Escherichia coli is a prolyl isomerase and a chaperone. It interacts with the ribosome and affects the folding of newly formed protein chains. Therefore, the dynamics of the interactions of trigger factor with the ribosome and with unfolded protein chains should be tailored for this function. Previously, we had found that binding of unfolded proteins to trigger factor is fast and that the lifetime of the complex between these two components is only about 100 ms. Here, we have labeled the trigger factor in its amino-terminal, ribosome-binding domain with a fluorescent dye and investigated how it interacts with the ribosome. We found that this association, as well as the dissociation of the complex, are rather slow processes. The average lifetime of the complex is about 30 seconds (at 20 degrees C). The strong differences in the dynamics of the interactions of trigger factor with the ribosome and with protein substrates might ensure that, on the one hand, trigger factor remains bound to the ribosome while a protein chain is being synthesized, and, on the other hand, allows it to scan the newly formed protein for prolyl bonds that need catalysis of isomerization.