In vitro delivery of the diphtheria toxin catalytic (C) domain from the lumen of purified early endosomes to the external milieu requires the addition of both ATP and a cytosolic translocation factor (CTF) complex. Using the translocation of C-domain ADP-ribosyltransferase activity across the endosomal membrane as an assay, the CTF complex activity was 650–800-fold purified from human T cell and yeast extracts, respectively. The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast. Further analysis of the role played by these two proteins with specific inhibitors, both in the in vitro translocation assay and in intact cell toxicity assays, has demonstrated their essential role in the productive delivery of the C-domain from the lumen of early endosomes to the external milieu. These results confirm and extend earlier observations of diphtheria toxin C-domain unfolding and refolding that must occur before and after vesicle membrane translocation. In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain. Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.

Precursor proteins of the acquired enamel pellicle derive from glandular and non-glandular secretions, which are components of whole saliva. The purpose of this investigation was to gain further insights into the characteristics of proteins in whole saliva and in vivo formed pellicle components. To maximize separation and resolution using only micro-amounts of protein, a two-dimensional gel electrophoresis system was employed. Protein samples from parotid secretion, submandibular/sublingual secretion, whole saliva, and pellicle were subjected to isoelectric focusing followed by SDS-PAGE. Selected protein spots were excised, subjected to "in-gel" trypsin digestion, and examined by mass spectrometry (MS). The data generated, including peptide maps and tandem MS spectra, were analyzed using protein data base searches. Components identified in whole saliva include cystatins (SA-III, SA, and SN), statherin, albumin, amylase, and calgranulin A. Components identified in pellicle included histatins, lysozyme, statherin, cytokeratins, and calgranulin B. The results showed that whole saliva and pellicle have more complex protein patterns than those of glandular secretions. There are some similarities and also distinct differences between the patterns of proteins present in whole saliva and pellicle. MS approaches allowed identification of not only well characterized salivary proteins but also novel proteins not previously identified in pellicle.Human teeth are exposed to whole saliva (WS), 1 consisting mainly of secretions derived from three pairs of major salivary glands, which comprise parotid, submandibular, and sublingual glands. Protein components that have been identified in all of the major glandular secretions are proline-rich proteins (acidic, basic, and glycosylated families), amylase, statherin, histatins, lysozyme, lactoferrin, lactoperoxidase, and secretory IgA (1-10), whereas cystatins and mucins have been identified in submandibular/sublingual secretions (9, 11-13). However, detailed understanding of the protein composition in WS is still limited because of the lack of knowledge about proteins in other contributors to whole saliva such as secretions from minor salivary glands and gingival crevicular fluid. In addition, little is known about modifications that occur on proteins during or after secretion into the oral cavity.The acquired enamel pellicle (EP) is a protein film thought to result from the selective adsorption of precursor proteins present in WS onto tooth surfaces. Because of its intimate contact with enamel surfaces, the EP plays an important role in maintaining tooth integrity by controlling the mineral solution dynamics of enamel. At its interface with the oral environment, the EP exerts selectivity on bacterial attachment and is involved in the initial stages of plaque formation (14). Because of the limiting amount of proteins that can be harvested from EP formed in vivo, previous investigations have utilized sensitive but indirect approaches such as enzymatic assays and immunologic detectio...

Hepatitis B viruses are pararetroviruses that contain a partially dsDNA genome and replicate this DNA through an RNA intermediate (the pregenomic RNA, pgRNA) by reverse transcription. Viral assembly begins with the packaging of the pgRNA into nucleocapsids (NCs), with subsequent reverse transcription within NCs converting the pgRNA into the characteristic dsDNA genome. Only NCs containing this dsDNA (the so-called ''mature'' NCs) are enveloped by the viral envelope proteins and secreted as virions; ''immature'' NCs, i.e., those containing pgRNA or immature reverse transcription intermediates, are excluded from virion formation. This phenomenon is thought to be caused by the emergence of an intrinsic maturation signal only on the mature NCs. To define the maturation signal, we have devised a method to separate mature from immature duck hepatitis B virus NCs and have compared them to NCs derived from secreted virions. Detailed mass spectrometric analyses revealed that the core protein from immature NCs was phosphorylated on at least six sites, whereas the core protein from mature NCs and that from secreted virions was entirely dephosphorylated. These results, together with the known requirement of core phosphorylation for pgRNA packaging and DNA synthesis, suggest that the NC undergoes a dynamic change in phosphorylation state to fulfill its multiple roles at different stages of viral replication. Although phosphorylation of the NCs is required for efficient RNA packaging and DNA synthesis by the immature NCs, dephosphorylation of the mature NCs may trigger envelopment and secretion.hepatitis B virus ͉ viral maturation ͉ posttranslational modification ͉ tandem mass spectrometry ͉ vibrational cooling MALDI-Fourier transform MS H epatitis B viruses (HBVs), or hepadnaviruses, are pararetroviruses that replicate their DNA genome via an RNA intermediate (pregenomic RNA, pgRNA) by reverse transcription (1, 2). Similar to classical retroviruses, hepadnaviruses begin assembly with the formation of intracellular nucleocapsid (NC) particles that specifically package viral pgRNA. However, they undergo reverse transcription before membrane envelopment of NCs and secretion as virions, rather than subsequent to entry into new target cells, like classical retroviruses (2, 3). This replication strategy is manifested in the hepadnaviral maturation phenomenon: secreted hepadnaviral virions contain only the mature, double-stranded relaxed circular (or the mature, doublestranded linear) DNA species, whereas intracellular pools of NCs contain a mix of nucleic acid species, including the pgRNA and DNA species from all of the intermediate stages of reverse transcription (2).The maturation phenomenon implies that mature DNAcontaining NCs may be selectively enveloped and secreted. It was proposed that DNA synthesis and NC envelopment may be coupled through a maturation signal (2). Strong support for this maturation signal model has been obtained (4-6), particularly through the use of a synchronized duck HBV (DHBV) replication system (7,8)...

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