No abstract

The major Euglena thylakoid protein, the light harvesting chlorophyll a/b-binding protein of photosystem II (pLHCPII) is synthesized in the cytoplasm as a polyprotein precursor composed of a 141 amino acid presequence containing a signal peptide domain followed by eight mature LHCPIIs covalently linked by a decapeptide. To determine the transport route from cytoplasm to chloroplast and the site of polyprotein processing, Euglena was pulse labeled with [35S]sulfate, organelles separated on sucrose gradients, and pLHCPII and LHCPII immunoprecipitated and separated on SDS gels. After a 10-min pulse, the pLHCPII polyprotein was found in the endoplasmic reticulum (ER) and Golgi apparatus. LHCPII was undetectable after a 10-min pulse consistent with the 20-min half-life for pLHCPII processing. When pulse-labeled cells were chased for 20 or 40 min with unlabeled sulfate, the fraction of pLHCPII in the ER decreased, and the fraction in the Golgi apparatus increased. LHCPII appeared only in thylakoids and chloroplasts, never in the ER or Golgi apparatus. Na2CO3 extraction, a treatment that releases soluble but not integral membrane proteins, did not remove pLHCPII from ER and Golgi membranes. Trypsin digestion of ER and Golgi membranes produced 4 pLHCPII membrane protected fragments. The Euglena pLHCPII polyprotein is transported as an integral membrane protein from the ER to the Golgi apparatus and from the Golgi apparatus to the chloroplast. Polyprotein processing appears to occur during or soon after chloroplast import of the membrane-bound precursor.

Identifying the structures that contribute to monoclonal antibody (mAb) binding sites (epitopes) within native G protein-coupled receptors (GPCRs) can be useful for developing topological models of the accessible receptor surface, for selecting the most relevant mAbs for therapeutic, diagnostic, and research applications and for distinguishing the intellectual property positions of otherwise similar mAbs. While conventional site-directed mutagenesis studies can identify individual amino acid residues that are critical to mAb binding, defining comprehensive epitopes is difficult and time-consuming for these structurally complex proteins. For example, in studies over the past decade, 13 residues (cumulatively) in the GPCR CCR5 have been reported to contribute to the interactions of five well-studied mAbs. 1-5 However, crystallographically defined epitopes contain an average of 20 contact residues each, 6 so these 13 residues likely represent only a portion of all the amino acids that constitute these five epitopes. Because of the importance of CCR5 in HIV infection and inflammation,7 more mAbs have been raised against the native form of this receptor than most other GPCRs, providing a useful set of tools to map its immunodominant structural regions. Here, we have used a high-throughput structure-function analysis strategy, which we refer to as "shotgun mutagenesis", to comprehensively map the critical residues, and in some cases the critical atoms, for these five epitopes of CCR5.To map mAb epitopes, we used an arrayed library of mutations covering nearly all the amino acids in the protein to identify amino acid changes that resulted in loss of mAb reactivity. This approach enabled each epitope to be rapidly mapped within a period of weeks. To create the mutant library, a parental CCR5 plasmid was first created, containing the full length (1059 bp) cDNA for wild type CCR5, flanked by a N-terminal HA epitope tag and a C-terminal V5 epitope tag. Cellular expression of the wild type tagged construct was confirmed by Western blot, immunofluorescence, and flow cytometry. Random mutations were next introduced into the parental CCR5 cDNA using a PCR-based method (Diversify PCR Random Mutagenesis kit, Clontech). Sequenced clones, most exhibiting one to two substitutions, were then selected from these random mutants to create a library with substitutions spanning the entire protein.The final library comprised 734 mutant CCR5 plasmids with substitutions in 346 of the 352 residues of CCR5 (>98% coverage). The average mutation rate per clone was 1.86 amino acids, and each amino acid position was substituted multiple times (an average of 3.95) across the entire library.We used this selective library of CCR5 mutants to map the epitopes of the anti-CCR5 mAbs CTC8, 45523, 45529, 45533 (R&D Systems), and 2D7 (Becton Dickinson). All five mAbs were originally derived, in three independent immunizations, by injecting mice with cells transiently overexpressing human CCR5. 4, 8 These mAbs are therefore representative of the murine immu...

The insulin-responsive 12-transmembrane transporter GLUT4 changes conformation between an inward-open state and an outward-open state to actively facilitate cellular glucose uptake. Because of the difficulties of generating conformational mAbs against complex and highly conserved membrane proteins, no reliable tools exist to measure GLUT4 at the cell surface, follow its trafficking, or detect the conformational state of the protein. Here we report the isolation and characterization of conformational mAbs that recognize the extracellular and intracellular domains of GLUT4, including mAbs that are specific for the inward-open and outward-open states of GLUT4. mAbs against GLUT4 were generated using virus-like particles to present this complex membrane protein in its native conformation and using a divergent host species (chicken) for immunization to overcome immune tolerance. As a result, the isolated mAbs recognize conformational epitopes on native GLUT4 in cells, with apparent affinities as high as 1 pM and with specificity for GLUT4 across the human membrane proteome. Epitope mapping using shotgun mutagenesis alanine scanning across the 509 amino acids of GLUT4 identified the binding epitopes for mAbs specific for the states of GLUT4 and allowed the comprehensive identification of the residues that functionally control the GLUT4 inward-open and outward-open states. The mAbs identified here will be valuable molecular tools for monitoring GLUT4 structure, function, and trafficking, for differentiating GLUT4 conformational states, and for the development of novel therapeutics for the treatment of diabetes.

Although bitter taste receptors (TAS2Rs) are important for human health, little is known of the determinants of ligand specificity. TAS2Rs such as TAS2R16 help define gustatory perception and dietary preferences that ultimately influence human health and disease. Each TAS2R must accommodate a broad diversity of chemical structures while simultaneously achieving high specificity so that diverse bitter toxins can be detected without all foods tasting bitter. However, how these G protein-coupled receptors achieve this balance is poorly understood. Here we used a comprehensive mutation library of human TAS2R16 to map its interactions with existing and novel agonists. We identified 13 TAS2R16 residues that contribute to ligand specificity and 38 residues whose mutation eliminated signal transduction by all ligands, providing a comprehensive assessment of how this GPCR binds and signals. Our data suggest a model in which hydrophobic residues on TM3 and TM7 form a broad ligand-binding pocket that can accommodate the diverse structural features of β-glycoside ligands while still achieving high specificity.