Benjamin Abell

Glycoprotein folding in the endoplasmic reticulum: A tale of three chaperones?

The endoplasmic reticulum (ER) is a major site of protein synthesis and its inside, or lumen, is a major site of protein folding. The lumen of the ER contains many folding factors and molecular chaperones, which facilitate protein folding by increasing both the rate and the efficiency of this process. Amongst the many ER folding factors, there are three components that specifically modulate the folding glycoproteins bearing N‐linked carbohydrate side chains. These components are calnexin, calreticulin and ERp57, and this review focuses on the molecular basis for their capacity to influence glycoprotein folding.

Signal recognition particle mediates post‐translational targeting in eukaryotes

Signal recognition particle (SRP) plays a central role in the delivery of classical secretory and membrane proteins to the endoplasmic reticulum (ER). All nascent chains studied to date dissociate from SRP once released from the ribosome, thereby supporting a strictly cotranslational mode of action for eukaryotic SRP. We now report a novel post‐translational function for SRP in the targeting of tail‐anchored (TA) proteins to the ER. TA proteins possess a hydrophobic membrane insertion sequence at their C‐terminus such that it can only emerge from the ribosome after translation is terminated. We show that SRP can associate post‐translationally with this type of ER‐targeting signal, and deliver newly synthesised TA proteins to the ER membrane by a pathway dependent upon GTP and the SRP receptor. We find that dependency upon this SRP‐dependent route is precursor specific, and propose a unifying model to describe the biogenesis of TA proteins in vivo.

Post-translational integration of tail-anchored proteins is facilitated by defined molecular chaperones

Tail-anchored (TA) proteins provide an ideal model for studying post-translational integration at the endoplasmic reticulum (ER) of eukaryotes. There are multiple pathways for delivering TA proteins from the cytosol to the ER membrane yet, whereas an ATP-dependent route predominates, none of the cytosolic components involved had been identified. In this study we have directly addressed this issue and identify novel interactions between a model TA protein and the two cytosolic chaperones Hsp40 and Hsc70. To investigate their function, we have reconstituted the membrane integration of TA proteins using purified components. Remarkably, we find that a combination of Hsc70 and Hsp40 can completely substitute for the ATP-dependent factors present in cytosol. On the basis of this in vitro analysis, we conclude that this chaperone pair can efficiently facilitate the ATP-dependent integration of TA proteins.

Alternative splicing of the auxin biosynthesis gene YUCCA4 determines its subcellular compartmentation.

Auxin is a major growth hormone in plants, and recent studies have elucidated many of the molecular mechanisms underlying its action, including transport, perception and signal transduction. However, major gaps remain in our knowledge of auxin biosynthetic control, partly due to the complexity and probable redundancy of multiple pathways that involve the YUCCA family of flavin-dependent mono-oxygenases. This study reveals the differential localization of YUCCA4 alternative splice variants to the endoplasmic reticulum and the cytosol, which depends on tissue-specific splicing. One isoform is restricted to flowers, and is anchored to the cytosolic face of the endoplasmic reticulum membrane via a hydrophobic C-terminal transmembrane domain. The other isoform is present in all tissues and is distributed throughout the cytosol. These findings are consistent with previous observations of yucca4 phenotypes in flowers, and suggest a role for intracellular compartmentation in auxin biosynthesis.

Subcellular distribution of tail-anchored proteins in Arabidopsis

Tail‐anchored (TA) proteins function in key cellular processes in eukaryotic cells, such as vesicle trafficking, protein translocation and regulation of transcription. They anchor to internal cell membranes by a C‐terminal transmembrane domain, which also serves as a targeting sequence. Targeting occurs post‐translationally, via pathways that are specific to the precursor, which makes TA proteins a model system for investigating post‐translational protein targeting. Bioinformatics approaches have previously been used to identify potential TA proteins in yeast and humans, yet little is known about TA proteins in plants. The identification of plant TA proteins is important for extending the post‐translational model system to plastids, in addition to general proteome characterization, and the identification of functional homologues characterized in other organisms. We identified 454 loci that potentially encode TA proteins in Arabidopsis, and combined published data with new localization experiments to assign localizations to 130 proteins, including 29 associated with plastids. By analysing the tail anchor sequences of characterized proteins, we have developed a tool for predicting localization and estimate that 138 TA proteins are localized to plastids.

Tail-anchored protein biosynthesis at the endoplasmic reticulum: the same but different

The post-translational integration of tail-anchored proteins at the endoplasmic reticulum represents a novel and distinct pathway for membrane protein synthesis. Studies of various precursors, exemplified by the synaptobrevins and cytochrome b5, indicate that multiple routes may facilitate their biosynthesis. There is clear evidence that both cytosolic factors and membrane components facilitate the efficient membrane insertion of at least some tail-anchored proteins. However, the nature of these mediators is currently unknown and their identification will be an essential step in defining the molecular basis of tail-anchored protein biogenesis.

Chaperone receptors: guiding proteins to intracellular compartments

Despite mitochondria and chloroplasts having their own genome, 99% of mitochondrial proteins (Rehling et al., Nat Rev Mol Cell Biol 5:519–530, 2004) and more than 95% of chloroplast proteins (Soll, Curr Opin Plant Biol 5:529–535, 2002) are encoded by nuclear DNA, synthesised in the cytosol and imported post-translationally. Protein targeting to these organelles depends on cytosolic targeting factors, which bind to the precursor, and then interact with membrane receptors to deliver the precursor into a translocase. The molecular chaperones Hsp70 and Hsp90 have been widely implicated in protein targeting to mitochondria and chloroplasts, and receptors capable of recognising these chaperones have been identified at the surface of both these organelles (Schlegel et al., Mol Biol Evol 24:2763–2774, 2007). The role of these chaperone receptors is not fully understood, but they have been shown to increase the efficiency of protein targeting (Young et al., Cell 112:41–50, 2003; Qbadou et al., EMBO J 25:1836–1847, 2006). Whether these receptors contribute to the specificity of targeting is less clear. A class of chaperone receptors bearing tetratricopeptide repeat domains is able to specifically bind the highly conserved C terminus of Hsp70 and/or Hsp90. Interestingly, at least of one these chaperone receptors can be found on each organelle (Schlegel et al., Mol Biol Evol 24:2763–2774, 2007), which suggests a universal role in protein targeting for these chaperone receptors. This review will investigate the role that chaperone receptors play in targeting efficiency and specificity, as well as examining recent in silico approaches to find novel chaperone receptors.

Tail-anchored membrane proteins: exploring the complex diversity of tail-anchored-protein targeting in plant cells.

Tail-anchored (TA) proteins are special class of integral membrane proteins that in recent years have received a considerable amount of attention due to their diverse cellular functions and unique targeting and insertion mechanisms. Defined by the presence of a single, hydrophobic membrane-spanning domain at or near their C terminus, TA proteins must be inserted into membranes post-translationally and are orientated such that their larger N-terminal domain (most often the functional domain) faces the cytosol, while their shorter C-terminal domain faces the interior of the organelle. The C-terminal domain of TA proteins also usually contains the information responsible for their selective targeting to the proper subcellular membrane, a process that, based primarily on studies with yeasts and mammals, appears to be highly complex due to the presence of multiple pathways. Within this context, we discuss here the biogenesis of plant TA proteins and the potential for hundreds of new TA proteins identified via bioinformatics screens to contribute to the already remarkable number of roles that this class of membrane proteins participates in throughout plant growth and development.

Study of receptor-chaperone interactions using the optical technique of spectroscopic ellipsometry

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms.

Quantification of Ligand Binding to G-Protein Coupled Receptors on Cell Membranes by Ellipsometry

G-protein-coupled receptors (GPCRs) are prime drug targets and targeted by approximately 60% of current therapeutic drugs such as β-blockers, antipsychotics and analgesics. However, no biophysical methods are available to quantify their interactions with ligand binding in a native environment. Here, we use ellipsometry to quantify specific interactions of receptors within native cell membranes. As a model system, the GPCR-ligand CXCL12α and its receptor CXCR4 are used. Human-derived Ishikawa cells were deposited onto gold coated slides via Langmuir-Schaefer film deposition and interactions between the receptor CXCR4 on these cells and its ligand CXCL12α were detected via total internal reflection ellipsometry (TIRE). This interaction could be inhibited by application of the CXCR4-binding drug AMD3100. Advantages of this approach are that it allows measurement of interactions in a lipid environment without the need for labelling, protein purification or reconstitution of membrane proteins. This technique is potentially applicable to a wide variety of cell types and their membrane receptors, providing a novel method to determine ligand or drug interactions targeting GPCRs and other membrane proteins.