We have used the homobifunctional cross-linking reagent disuccinimidyl suberate (DSS) to identify proteins that are adjacent to nascent polypeptides undergoing translocations across mammalian rough ER. Translocation intermediates were assembled by supplementing cell free translations of truncated mRNAs with the signal recognition particle (SRP) and microsomal membrane vesicles. Two prominent cr...

Lipids and proteins were found to contact a nascent type II membrane protein, as well as a nascent secretory protein, during their insertion into the membrane of the endoplasmic reticulum. This suggests that the protein-conducting channel is open laterally toward the lipid bilayer during an early stage of protein insertion. Contact to lipids was confined to the hydrophobic core region of the re...

The eubacterial chaperonins GroEL and GroES are essential chaperones and primarily assist protein folding in the cell. Although the molecular mechanism of the GroEL system has been examined previously, the mechanism by which GroEL and GroES assist folding of nascent polypeptides during translation is still poorly understood. We previously demonstrated a co-translational involvement of the Esche...

De novo protein folding is delicate and error-prone and requires the guidance of molecular chaperones. Besides cytosolic and organelle-specific chaperones, cells have evolved ribosome-associated chaperones that support early folding events and prevent misfolding and aggregation. This class of chaperones includes the bacterial trigger factor (TF), the archaeal and eukaryotic nascent polypeptide-...

All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes ...

We have previously identified the transcript encoding NACA (the alpha chain of the nascent-polypeptide-associated complex) as a cytokine-modulated specific transcript in the human TF-1 erythroleukemic cell line. This protein was already known to be a transcriptional co-activator that acts by potentiating AP-1 activity in osteoblasts, and is known to be involved in the targeting of nascent polyp...

In all organisms, the ribosome synthesizes and folds full length polypeptide chains into active three-dimensional conformations. The nascent protein goes through two major interactions, first with the ribosome which synthesizes the polypeptide chain and holds it for a considerable length of time, and then with the chaperones. Some of the chaperones are found in solution as well as associated to...

Cotranslational degradation of polypeptide nascent chains plays a critical role in quality control of protein synthesis and the rescue of stalled ribosomes. In eukaryotes, ribosome stalling triggers release of 60S subunits with attached nascent polypeptides, which undergo ubiquitination by the E3 ligase Ltn1 and proteasomal degradation facilitated by the ATPase Cdc48. However, the identity of f...