Arthur E. Johnson

N-Linked Glycans Direct the Cotranslational Folding Pathway of Influenza Hemagglutinin

For proteins that traverse the secretory pathway, folding commences cotranslationally upon translocation into the endoplasmic reticulum. In this study, we have comprehensively analyzed the earliest maturation steps of the model glycoprotein influenza hemagglutinin (HA). These steps include cleavage of the signal sequence, glycosylation, binding by the chaperones calnexin and calreticulin, and the oxidoreductase ERp57, and oxidation. Our results show that the molecular choreography of the nascent HA chain is largely directed by multiple glycans that are strategically placed to elicit the binding of lectin chaperones. These chaperones are recruited to specific nascent chain locations to regulate and facilitate glycoprotein folding, thereby suggesting that the positioning of N-linked glycans in critical regions has evolved to optimize the folding process in the cell.

Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex

In eukaryotic cells, polypeptides are N glycosylated after passing through the membrane of the ER into the ER lumen. This modification is effected cotranslationally by the multimeric oligosaccharyltransferase (OST) enzyme. Here, we report the first cross-linking of an OST subunit to a nascent chain that is undergoing translocation through, or integration into, the ER membrane. A photoreactive probe was incorporated into a nascent chain using a modified Lys-tRNA and was positioned in a cryptic glycosylation site (-Q-K-T- instead of -N-K-T-) in the nascent chain. When translocation intermediates with nascent chains of increasing length were irradiated, nascent chain photocross-linking to translocon components, Sec61α and TRAM, was replaced by efficient photocross-linking solely to a protein identified by immunoprecipitation as the STT3 subunit of the OST. No cross-linking was observed in the absence of a cryptic sequence or in the presence of a competitive peptide substrate of the OST. As no significant nascent chain photocross-linking to other OST subunits was detected in these fully assembled translocation and integration intermediates, our results strongly indicate that the nascent chain portion of the OST active site is located in STT3.

A new role for BiP closing the aqueous translocon pore during protein integration into the ER membrane

In mammalian cells, most membrane proteins are inserted cotranslationally into the ER membrane at sites termed translocons. Although each translocon forms an aqueous pore, the permeability barrier of the membrane is maintained during integration, even when the otherwise tight ribosome–translocon seal is opened to allow the cytoplasmic domain of a nascent protein to enter the cytosol. To identify the mechanism by which membrane integrity is preserved, nascent chain exposure to each side of the membrane was determined at different stages of integration by collisional quenching of a fluorescent probe in the nascent chain. Comparing integration intermediates prepared with intact, empty, or BiP-loaded microsomes revealed that the lumenal end of the translocon pore is closed by BiP in an ATP-dependent process before the opening of the cytoplasmic ribosome–translocon seal during integration. This BiP function is distinct from its previously identified role in closing ribosome-free, empty translocons because of the presence of the ribosome at the translocon and the nascent membrane protein that extends through the translocon pore and into the lumen during integration. Therefore, BiP is a key component in a sophisticated mechanism that selectively closes the lumenal end of some, but not all, translocons occupied by a nascent chain. By using collisional quenchers of different sizes, the large internal diameter of the ribosome-bound aqueous translocon pore was found to contract when BiP was required to seal the pore during integration. Therefore, closure of the pore involves substantial conformational changes in the translocon that are coupled to a complex sequence of structural rearrangements on both sides of the ER membrane involving the ribosome and BiP.

SRP Keeps Polypeptides Translocation-Competent by Slowing Translation to Match Limiting ER-Targeting Sites

SRP is essential for targeting nascent chains to the endoplasmic reticulum, and it delays nascent chain elongation in cell-free translation systems. However, the significance of this function has remained unclear. We show that efficient protein translocation into the ER is incompatible with normal cellular translation rates due to rate-limiting concentrations of SRP receptor (SR). We complemented mammalian cells depleted of SRP14 by expressing mutant versions of the protein lacking the elongation arrest function. The absence of a delay caused inefficient targeting of preproteins leading to defects in secretion, depletion of proteins in the endogenous membranes, and reduced cell growth. The detrimental effects were reversed by either reducing the cellular protein synthesis rate or increasing SR expression. SRP therefore ensures that nascent chains remain translocation competent during the targeting time window dictated by SR. Since SRP-signal sequence affinities vary, the delay may also regulate which proteins are preferentially targeted.

Fluorescence approaches for determining protein conformations, interactions and mechanisms at membranes.

Processes that occur at membranes are essential for the viability of every cell, but such processes are the least well understood at the molecular level. The complex nature and physical properties of the molecular components involved, as well as the requirement for two separated aqueous compartments, restrict the experimental approaches that can be successfully applied to examine the structure, conformational changes and interactions of the membrane‐bound proteins that accomplish these processes. In particular, to accurately elucidate the molecular mechanisms that effect and regulate such processes, one must use experimental approaches that do not disrupt the structural integrity or functionality of the protein–membrane complexes being examined. To best accomplish this goal, especially when large multicomponent complexes and native membranes are involved, the optimal experimental approach to use is most often fluorescence spectroscopy. Using multiple independent fluorescence techniques, one can determine structural information in real time and in intact membranes under native conditions that cannot be obtained by crystallography, electron microscopy and NMR techniques, among others. Furthermore, fluorescence techniques provide a comprehensive range of information, from kinetic to thermodynamic, about the assembly, structure, function and regulation of membrane‐bound proteins and complexes. This article describes the use of various fluorescence techniques to characterize different aspects of proteins bound to or embedded in membranes.

Pore-forming protein structure analysis in membranes using multiple independent fluorescence techniques

A large number of transmembrane proteins form aqueous pores or channels in the phospholipid bilayer, but the structural bases of pore formation and assembly have been determined experimentally for only a few of the proteins and protein complexes. The polypeptide segments that form the transmembrane pore and the secondary structure that creates the aqueous-lipid interface can be identified using multiple independent fluorescence techniques (MIFT). The information obtained from several different, but complementary, fluorescence analyses, including measurements of emission intensity, fluorescence lifetime, accessibility to aqueous and to lipophilic quenching agents, and fluorescence resonance energy transfer (FRET) can be combined to characterize the nature of the protein-membrane interaction directly and unambiguously. The assembly pathway can also be determined by measuring the kinetics of the spectral changes that occur upon pore formation. The MIFT approach therefore allows one to obtain structural information that cannot be obtained easily using alternative techniques such as crystallography. This review briefly outlines how MIFT can reveal the identity, location, conformation, and topography of the polypeptide sequences that interact with the membrane.

Protein Sorting at the Membrane of the Endoplasmic Reticulum

Publisher Summary This chapter provides a brief discussion on protein sorting at the membrane of the endoplasmic reticulum. After targeting, the cell often must direct the facilitated transport of polypeptides across a membrane bilayer. The focus of this chapter is on the protein translocation across the membrane of the endoplasmic reticulum (ER), the first step in the cellular secretory pathway in eukaryotic cells. The signal recognition particle (SRP) is a ribonucleoprotein complex that is responsible for signal sequence recognition. In post-translational translocation, a secretory protein is completely synthesized by the ribosome in the cytoplasm before the translocation process begins. Independent confirmation that the nascent chain occupies an aqueous pore through the membrane during translocation was obtained using iodide ions as hydrophilic collisional quenchers of fluorescence. As a more complete picture of the various functions performed by the translocon emerges, global issues of regulation and cross-talk between functional pathways that use the translocon machinery will be of further interest.