Johanna Dudek

Functions and pathologies of BiP and its interaction partners

Abstract.The endoplasmic reticulum (ER) is involved in a variety of essential and interconnected processes in human cells, including protein biogenesis, signal transduction, and calcium homeostasis. The central player in all these processes is the ER-lumenal polypeptide chain binding protein BiP that acts as a molecular chaperone. BiP belongs to the heat shock protein 70 (Hsp70) family and crucially depends on a number of interaction partners, including co-chaperones, nucleotide exchange factors, and signaling molecules. In the course of the last five years, several diseases have been linked to BiP and its interaction partners, such as a group of infectious diseases that are caused by Shigella toxin producing E. coli. Furthermore, the inherited diseases Marinesco-Sjögren syndrome, autosomal dominant polycystic liver disease, Wolcott-Rallison syndrome, and several cancer types can be considered BiP-related diseases. This review summarizes the physiological and pathophysiological characteristics of BiP and its interaction partners.

BiP‐mediated closing of the Sec61 channel limits Ca2+ leakage from the ER

In mammalian cells, signal peptide‐dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic protein‐conducting channel, the Sec61 complex. Previous work has characterized the Sec61 channel as a potential ER Ca2+ leak channel and identified calmodulin as limiting Ca2+ leakage in a Ca2+‐dependent manner by binding to an IQ motif in the cytosolic aminoterminus of Sec61α. Here, we manipulated the concentration of the ER lumenal chaperone BiP in cells in different ways and used live cell Ca2+ imaging to monitor the effects of reduced levels of BiP on ER Ca2+ leakage. Regardless of how the BiP concentration was lowered, the absence of available BiP led to increased Ca2+ leakage via the Sec61 complex. When we replaced wild‐type Sec61α with mutant Sec61αY344H in the same model cell, however, Ca2+ leakage from the ER increased and was no longer affected by manipulation of the BiP concentration. Thus, BiP limits ER Ca2+ leakage through the Sec61 complex by binding to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344.

Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon

In mammalian cells, proteins are typically translocated across the endoplasmic reticulum (ER) membrane in a co-translational mode by the ER protein translocon, comprising the protein-conducting channel Sec61 and additional complexes involved in nascent chain processing and translocation. As an integral component of the translocon, the oligosaccharyl-transferase complex (OST) catalyses co-translational N-glycosylation, one of the most common protein modifications in eukaryotic cells. Here we use cryoelectron tomography, cryoelectron microscopy single-particle analysis and small interfering RNA-mediated gene silencing to determine the overall structure, oligomeric state and position of OST in the native ER protein translocon of mammalian cells in unprecedented detail. The observed positioning of OST in close proximity to Sec61 provides a basis for understanding how protein translocation into the ER and glycosylation of nascent proteins are structurally coupled. The overall spatial organization of the native translocon, as determined here, serves as a reliable framework for further hypothesis-driven studies.

Different effects of Sec61α, Sec62 and Sec63 depletion on transport of polypeptides into the endoplasmic reticulum of mammalian cells

Co-translational transport of polypeptides into the endoplasmic reticulum (ER) involves the Sec61 channel and additional components such as the ER lumenal Hsp70 BiP and its membrane-resident co-chaperone Sec63p in yeast. We investigated whether silencing the SEC61A1 gene in human cells affects co- and post-translational transport of presecretory proteins into the ER and post-translational membrane integration of tail-anchored proteins. Although silencing the SEC61A1 gene in HeLa cells inhibited co- and post-translational transport of signal-peptide-containing precursor proteins into the ER of semi-permeabilized cells, silencing the SEC61A1 gene did not affect transport of various types of tail-anchored protein. Furthermore, we demonstrated, with a similar knockdown approach, a precursor-specific involvement of mammalian Sec63 in the initial phase of co-translational protein transport into the ER. By contrast, silencing the SEC62 gene inhibited only post-translational transport of a signal-peptide-containing precursor protein.

ERj1p has a basic role in protein biogenesis at the endoplasmic reticulum

ERj1p is a membrane protein of the endoplasmic reticulum (ER) that can recruit the ER lumenal chaperone BiP to translating ribosomes. ERj1p can also modulate protein synthesis at initiation and is predicted to be a membrane-tethered transcription factor. Here we attribute the various functions of ERj1p to distinct regions within its cytosolic domain. A highly positively charged nonapeptide within this domain is necessary and sufficient for binding to ribosomes. Binding of ERj1p to ribosomes involves the 28S ribosomal RNA and occurs at the tunnel exit. Additionally, ERj1p has a dual regulatory role in gene expression: ERj1p inhibits translation in the absence of BiP, and another charged oligopeptide within the cytosolic domain of ERj1p mediates binding of the nuclear import factor importin β and import into the nucleus, thereby paving the way for subsequent action on genomic DNA.

Evolutionary Gain of Function for the ER Membrane Protein Sec62 from Yeast to Humans

We characterized interactions between the human proteins Sec62 and Sec63 as well as the putative interaction of human Sec62 with ribosomes. The data demonstrate evolutionary conservation of Sec62/Sec63 interaction and indicate that in the course of evolution Sec62 of vertebrates has gained the additional function to interact with ribosomes.

Interaction of calmodulin with Sec61α limits Ca2+ leakage from the endoplasmic reticulum

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein‐conducting channel, the Sec61 complex. The presence of large, water‐filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)‐binding motif in the cytosolic N‐terminus of mammalian Sec61α that bound CaM but not Ca2+‐free apocalmodulin with nanomolar affinity and sequence specificity. In single‐channel measurements, CaM potently mediated Sec61‐channel closure in Ca2+‐dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+‐CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER.

A novel type of co-chaperone mediates transmembrane recruitment of DnaK-like chaperones to ribosomes.

Recently, the homolog of yeast protein Sec63p was identified in dog pancreas microsomes. This pancreatic DnaJ‐like protein was shown to be an abundant protein, interacting with both the Sec61p complex and lumenal DnaK‐like proteins, such as BiP. The pancreatic endoplasmic reticulum contains a second DnaJ‐like membrane protein, which had been termed Mtj1p in mouse. Mtj1p is present in pancreatic microsomes at a lower concentration than Sec63p but has a higher affinity for BiP. In addition to a lumenal J‐domain, Mtj1p contains a single transmembrane domain and a cytosolic domain which is in close contact with translating ribosomes and appears to have the ability to modulate translation. The interaction with ribosomes involves a highly charged region within the cytosolic domain of Mtj1p. We propose that Mtj1p represents a novel type of co‐chaperone, mediating transmembrane recruitment of DnaK‐like chaperones to ribosomes and, possibly, transmembrane signaling between ribosomes and DnaK‐like chaperones of the endoplasmic reticulum.

The SND proteins constitute an alternative targeting route to the endoplasmic reticulum

In eukaryotes, up to one-third of cellular proteins are targeted to the endoplasmic reticulum, where they undergo folding, processing, sorting and trafficking to subsequent endomembrane compartments. Targeting to the endoplasmic reticulum has been shown to occur co-translationally by the signal recognition particle (SRP) pathway or post-translationally by the mammalian transmembrane recognition complex of 40 kDa (TRC40) and homologous yeast guided entry of tail-anchored proteins (GET) pathways. Despite the range of proteins that can be catered for by these two pathways, many proteins are still known to be independent of both SRP and GET, so there seems to be a critical need for an additional dedicated pathway for endoplasmic reticulum relay. We set out to uncover additional targeting proteins using unbiased high-content screening approaches. To this end, we performed a systematic visual screen using the yeast Saccharomyces cerevisiae, and uncovered three uncharacterized proteins whose loss affected targeting. We suggest that these proteins work together and demonstrate that they function in parallel with SRP and GET to target a broad range of substrates to the endoplasmic reticulum. The three proteins, which we name Snd1, Snd2 and Snd3 (for SRP-independent targeting), can synthetically compensate for the loss of both the SRP and GET pathways, and act as a backup targeting system. This explains why it has previously been difficult to demonstrate complete loss of targeting for some substrates. Our discovery thus puts in place an essential piece of the endoplasmic reticulum targeting puzzle, highlighting how the targeting apparatus of the eukaryotic cell is robust, interlinked and flexible.

The heat shock protein 70 molecular chaperone network in the pancreatic endoplasmic reticulum − a quantitative approach

Traditionally, the canine pancreatic endoplasmic reticulum (ER) has been the workhorse for cell‐free studies on protein transport into the mammalian ER. These studies have revealed multiple roles for the major ER‐luminal heat shock protein (Hsp) 70, IgG heavy chain‐binding protein (BiP), at least one of which also involves the second ER‐luminal Hsp70, glucose‐regulated protein (Grp) 170. In addition, at least one of these BiP activities depends on Hsp40. Up to now, five Hsp40s and two nucleotide exchange factors, Sil1 and Grp170, have been identified in the ER of different mammalian cell types. Here we quantified the various proteins of this chaperone network in canine pancreatic rough microsomes. We also characterized the various purified proteins with respect to their affinities for BiP and their effect on the ATPase activity of BiP. The results identify Grp170 as the major nucleotide exchange factor for BiP, and the resident ER‐membrane proteins ER‐resident J‐domain protein 1 plus ER‐resident J‐domain protein 2/Sec63 as prime candidates for cochaperones of BiP in protein transport in the pancreatic ER. Thus, these data represent a comprehensive analysis of the BiP chaperone network that was recently linked to two human inherited diseases, polycystic liver disease and Marinesco–Sjögren syndrome.