Defining the role of the Escherichia coli chaperone SecB using comparative proteomics

To improve understanding and identify novel substrates of the cytoplasmic chaperone SecB in Escherichia coli, we analyzed a secB null mutant using comparative proteomics. The secB null mutation did not affect cell growth but caused significant differences at the proteome level. In the absence of SecB, dynamic protein aggregates containing predominantly secretory proteins accumulated in the cytoplasm. Unprocessed secretory proteins were detected in radiolabeled whole cell lysates. Furthermore, the assembly of a large fraction of the outer membrane proteome was slowed down, whereas its steady state composition was hardly affected. In response to aggregation and delayed sorting of secretory proteins, cytoplasmic chaperones DnaK, GroEL/ES, ClpB, IbpA/B, and HslU were up-regulated severalfold, most likely to stabilize secretory proteins during their delayed translocation and/or rescue aggregated secretory proteins. The SecB/A dependence of 12 secretory proteins affected by the secB null mutation (DegP, FhuA, FkpA, OmpT, OmpX, OppA, TolB, TolC, YbgF, YcgK, YgiW, and YncE) was confirmed by "classical" pulse-labeling experiments. Our study more than triples the number of known SecB-dependent secretory proteins and shows that the primary role of SecB is to facilitate the targeting of secretory proteins to the Sec-translocase.     

Secretory Proteins from Adrenal Medullary Cells Are Carboxyl-Methylated In Vivo and Released Under Their Methylated Form by Acetylcholine

The carboxyl methylation of secretory proteins in vivo was investigated in bovine adrenal medullary cells in culture. Chromogranin A, the major intragranular secretory protein in adrenal medullary cells, and other secretory proteins were found to be carboxyl-methylated within secretory vesicles. The in vivo labeling pattern using [methyl-3H]methionine and the in vitro labeling pattern using S-adenosyl-[methyl-14C]methionine of intravesicular secretory proteins were similar. The detection of methylated chromogranin A in mature secretory vesicles required 3-6 h, a time consistent with the synthesis and storage of secretory proteins in this tissue. Carboxyl-methylated chromogranin A was secreted from medullary cells by exocytosis via activation of nicotinic cholinergic receptor and recovered still under the methylated form in the incubation medium. Since protein-carboxyl-methylase is cytosolic, these results suggest that methylation of secretory proteins is a cotranslational phenomenon.     

Isoproterenol increases sorting of parotid gland cargo proteins to the basolateral pathway

Exocrine cells have an essential function of sorting secreted proteins into the correct secretory pathway. A clear understanding of sorting in salivary glands would contribute to the correct targeting of therapeutic transgenes. The present work investigated whether there is a change in the relative proportions of basic proline-rich protein (PRP) and acidic PRPs in secretory granules in response to chronic isoproterenol treatment, and whether this alters the sorting of endogenous cargo proteins. Immunoblot analysis of secretory granules from rat parotids found a large increase of basic PRP over acidic PRPs in response to chronic isoproterenol treatment. Pulse chase experiments demonstrated that isoproterenol also decreased regulated secretion of newly synthesized secretory proteins, including PRPs, amylase and parotid secretory protein. This decreased efficiency of the apical regulated pathway may be mediated by alkalization of the secretory granules since it was reversed by treatment with mild acid. We also investigated changes in secretion through the basolateral (endocrine) pathways. A significant increase in parotid secretory protein and salivary amylase was detected in sera of isoproterenol-treated animals, suggesting increased routing of the regulated secretory proteins to the basolateral pathway. These studies demonstrate that shifts of endogenous proteins can modulate regulated secretion and sorting of cargo proteins. amylase; parotid secretory protein; polarized secretion     

Comparison of proteins expressed on secretory vesicle membranes and plasma membranes of human neutrophils

Secretory vesicles are neutrophil intracellular storage granules formed by endocytosis. Understanding the functional consequences of secretory vesicle exocytosis requires knowledge of their membrane proteins. The current study was designed to use proteomic technologies to develop a more complete catalog of secretory vesicle membrane proteins and to compare the proteomes of secretory vesicle and plasma membranes. A total of 1118 proteins were identified, 573 (51%) were present only in plasma membrane-enriched fractions, 418 (37%) only in secretory vesicle-enriched membrane fractions, and 127 (11%) in both fractions. Gene Ontology categorized 373 of these proteins as integral membrane proteins. Proteins typically associated with other intracellular organelles, including nuclei, mitochondria, and ribosomes, were identified in both membrane fractions. Ingenuity Pathway Knowledge Base analysis determined that the majority of canonical and functional pathways were significantly associated with proteins from both plasma membrane-enriched and secretory vesicle-enriched fractions. There were, however, some canonical signaling pathways that involved proteins only from plasma membranes or secretory vesicles. In conclusion, a number of proteins were identified that may elucidate mechanisms and functional consequences of secretory vesicle exocytosis. The small number of common proteins suggests that the hypothesis that secretory vesicles are formed from plasma membranes by endocytosis requires more critical evaluation.     

Armed and dangerous: Toxoplasma gondii uses an arsenal of secretory proteins to infect host cells

Toxoplasma gondii is a protozoan parasite that infects a wide variety of warm-blooded animals and humans, in which it causes opportunistic disease. As an obligate intracellular parasite, T. gondii must invade a host cell to survive and replicate during infection. Recent studies suggest that T. gondii secretes a variety of proteins that appear to function during invasion or intracellular replication. These proteins originate from three distinct regulated secretory organelles called micronemes, rhoptries and dense granules. By discharging the contents of its secretory organelles at precise steps in invasion, T. gondii appears to timely deploy secretory proteins to their correct target destinations. Based on the timing of secretion and the characteristics of secretory proteins, an emerging theme is that T. gondii compartmentalizes its secretory proteins according to general function. Thus, it appears that micronemal proteins may function during parasite attachment to host cells, rhoptry proteins may facilitate parasite vacuole formation and host organellar association, and dense granule proteins likely promote intracellular replication, possibly by transporting and processing nutrients from the host cell. However, as more T. gondii secretory proteins are identified and characterized, it is likely that additional functions will be ascribed to each class of proteins secreted- by this fascinating invasive parasite.     

Protein targeting to dense-core secretory granules

Regulated secretory proteins are stored within specialized vesicles known as secretory granules. It is not known how proteins are sorted into these organelles. Regulated proteins may possess targeting signals which interact with specific sorting receptors in the lumen of the trans-Golgi network (TGN) prior to their aggregation to form the characteristic dense-core of the granule. Alternatively, sorting may occur as the result of specific aggregation of regulated proteins in the TGN. Aggregates may be directed to secretory granules by interaction of a targeting signal on the surface with a sorting receptor. Novel targeting signals which confer on regulated proteins a tendency to aggregate under certain conditions, and in so doing cause them to be incorporated into secretory granules, have been implicated. Specific targeting signals may also play a role in directing membrane proteins to secretory granules.     

非经典的蛋白质分泌途径

蛋白质分泌是细胞间进行信息传递的重要方式之一。一般的分泌蛋白在其N端都具有信号肽序列,可以通过经典的内质网-高尔基体途径进行运输,并最终分泌到细胞外。近来发现细胞内存在另一类分泌蛋白,它们能够分泌到细胞外发挥功能,但却没有典型的信号肽序列,被称为非经典分泌蛋白。越来越多的证据表明,这类蛋白质的分泌有其独特的机制。本综述了这类蛋白质的各种分泌机制及可能的生理意义。     

The opportunistic pathogen Toxoplasma gondii deploys a diverse legion of invasion and survival proteins

Host cell invasion is an essential step during infection by Toxoplasma gondii, an intracellular protozoan that causes the severe opportunistic disease toxoplasmosis in humans. Recent evidence strongly suggests that proteins discharged from Toxoplasma apical secretory organelles (micronemes, dense granules, and rhoptries) play key roles in host cell invasion and survival during infection. However, to date, only a limited number of secretory proteins have been discovered, and the full spectrum of effector molecules involved in parasite invasion and survival remains unknown. To address these issues, we analyzed a large cohort of freely released Toxoplasma secretory proteins by using two complementary methodologies, two-dimensional electrophoresis/mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry (MudPIT, shotgun proteomics). Visualization of Toxoplasma secretory products by two-dimensional electrophoresis revealed approximately 100 spots, most of which were successfully identified by protein microsequencing or matrix-assisted laser desorption ionization-mass spectrometry analysis. Many proteins were present in multiple species suggesting they are subjected to substantial post-translational modification. Shotgun proteomic analysis of the secretory fraction revealed several additional products, including novel putative adhesive proteins, proteases, and hypothetical secretory proteins similar to products expressed by other related parasites including Plasmodium, the etiologic agent of malaria. A subset of novel proteins were re-expressed as fusions to yellow fluorescent protein, and this initial screen revealed shared and distinct localizations within secretory compartments of T. gondii tachyzoites. These findings provided a uniquely broad view of Toxoplasma secretory proteins that participate in parasite survival and pathogenesis during infection.     

Modulation of secretory granule-targeting efficiency by cis and trans compounding of sorting signals

Several protein domains acting through seemingly different mechanisms have been reported to have the capacity to target proteins to dense core secretory granules. Because proteins enter secretory granules with different efficiencies and because some of these proteins contain more than one granule-targeting motif, we have investigated whether compounding sorting signals could alter the efficiency of protein entry into secretory granules. In the current study we demonstrate that a paired basic cleavage site from human prorenin and an alpha-helix-containing secretory granule-sorting signal from the prohormone convertase PC1/3 can synergize to increase granule-sorting efficiency not only when located on the same protein, but also when located on distinct proteins that associate in the secretory pathway.     

Secretory cargo composition affects polarized secretion in MDCK epithelial cells

Polarized epithelial cells secrete proteins at either the apical or basolateral cell surface. A number of non-epithelial secretory proteins also exhibit polarized secretion when they are expressed in polarized epithelial cells but it is difficult to predict where foreign proteins will be secreted in epithelial cells. The question is of interest since secretory epithelia are considered as target tissues for gene therapy protocols that aim to express therapeutic secretory proteins. In the parathyroid gland, parathyroid hormone is processed by furin and co-stored with chromogranin A in secretory granules. To test the secretion of these proteins in epithelial cells, they were expressed in MDCK cells. Chromogranin A and a secreted form of furin were secreted apically while parathyroid hormone was secreted 60% basolaterally. However, in the presence of chromogranin A, the secretion of parathyroid hormone was 65% apical, suggesting that chromogranin can act as a “sorting escort” (sorting chaperone) for parathyroid hormone. Conversely, apically secreted furin did not affect the sorting of parathyroid hormone. The apical secretion of chromogranin A was dependent on cholesterol, suggesting that this protein uses an established cellular sorting mechanism for apical secretion. However, this sorting does not involve the N-terminal membrane-binding domain of chromogranin A. These results suggest that foreign secretory proteins can be used as “sorting escorts” to direct secretory proteins to the apical secretory pathway without altering the primary structure of the secreted protein. Such a system may be of use in the targeted expression of secretory proteins from epithelial cells. David V. Cohn—Deceased.     

A hydrophobic patch in a charged alpha-helix is sufficient to target proteins to dense core secretory granules

Many endocrine and neuroendocrine cells contain specialized secretory organelles called dense core secretory granules. These organelles are the repository of proteins and peptides that are secreted in a regulated manner when the cell receives a physiological stimulus. The targeting of proteins to these secretory granules is crucial for the generation of certain peptide hormones, including insulin and ACTH. Although previous work has demonstrated that proteins destined to a variety of cellular locations, including secretory granules, contain targeting sequences, no single consensus sequence for secretory granule-sorting signals has emerged. We have shown previously that alpha-helical domains in the C-terminal tail of the prohormone convertase PC1/3 play an important role in the ability of this region of the protein to direct secretory granule targeting (Jutras, I. Seidah, N. G., and Reudelhuber, T. L. (2000) J. Biol. Chem. 275, 40337-40343). In this study, we show that a variety of alpha-helical domains are capable of directing a heterologous secretory protein to granules. By testing a series of synthetic alpha-helices, we also demonstrate that the presence of charged (either positive or negative) amino acids spatially segregated from a hydrophobic patch in the alpha-helices of secretory proteins likely plays a critical role in the ability of these structures to direct secretory granule sorting.     

真核细胞非经典蛋白分泌途径

在生物体中, 细胞间的信息传递是细胞生长、分化、发育、增殖、凋亡等生命活动的基本保证, 而蛋白分泌是细胞间信息传递的重要方式。大多数分泌蛋白都是通过内质网-高尔基体(ER-Golgi)途径分泌的。然而越来越多的研究表明, 存在着一类无信号肽的分泌蛋白, 这类蛋白不依赖ER-Golgi途径就能分泌到细胞外发挥功能, 被称为非经典分泌蛋白。非经典蛋白的分泌有其特有的机制, 它对ER-Golgi分泌途径是一种必要和有益的补充。非经典分泌与细胞增殖、免疫反应、肿瘤形成、传染病病理学等密切相关。文章旨在对非经典分泌蛋白的特点、分泌机制及生物学意义进行概述。     

Regulated, but not constitutive, secretory proteins bind porcine chymotrypsinogen.

Endocrine and exocrine cells exhibit both a constitutive and a regulated secretory pathway. In the latter pathway, secretory proteins are stored at a high concentration in secretory granules and are released by exocytosis in response to appropriate external stimuli. Sorting between the two secretory pathways is believed to take place in the trans-Golgi tubular network. To account for experimental data, it has been proposed that sorting receptors exist which bind a variety of regulated secretory proteins, including foreign secretory proteins introduced into the cells by transfection. In support of the sorting receptor hypothesis Chung et al. (Chung, K.-N., Walter, P., Aponte, G. W., and Moore, H.-P.H. (1989) Science 243, 192-197) isolated a group of 25-kDa canine pancreatic "hormone-binding proteins" that bound regulated but not constitutive secretory proteins. To determine if similar proteins are present in other species and tissues, we have screened porcine pancreas, parathyroid, adrenal medulla, and pituitary glands. A 31-kDa protein, similar to that identified by Chung et al. (1989), which binds to regulated but not to constitutive secretory proteins was identified in porcine pancreas. This protein was not detected in the parathyroid, adrenal medulla, or pituitary glands, however, which argues against it serving as a general sorting receptor. NH2-terminal sequencing, immunoreactivity, and proteolytic activity data indicate that the porcine 31-kDa protein is similar if not identical to porcine chymotrypsinogen A or B.     

Export of proteins from oocytes of Xenopus laevis.

When human lymphoblastoid mRNA was microinjected into X. laevis oocytes, titers of interferon rapidly reached a maximum inside the oocyte while accumulation of interferon continued in the incubation medium for at least 45 hr. If interferon protein was injected into oocytes it was rapidly inactivated. Significantly, newly synthesized interferon but not injected interferon was found to be membrane-associated. Further experiments involving the co-injection of mRNAs coding for secretory proteins (guinea pig milk proteins and human interferon) and nonsecretory proteins (rabbit globin) revealed that only the secretory proteins were exported from the oocyte. Moreover, different proteins were exported at different rates. A distinct subclass of newly synthesized oocyte proteins of unknown function also accumulated in the incubation medium. Since the information encoded in the messenger RNAs of secretory proteins is sufficient to specify synthesis, compartmentation and secretion of these proteins, the oocyte may provide a complete system for the analysis of the secretory process.     

Differential aggregation properties of secretory proteins that are stored in exocrine secretory granules of the pancreas and parotid glands

Low-pH- and calcium-induced aggregation of regulated secretory proteins has been proposed to play a role in their retention and storage in secretory granules. However, this has not been tested for secretory proteins that are stored in the exocrine parotid secretory granules. Parotid granule matrix proteins were analyzed for aggregation in the presence or absence of calcium and in the pH range of 5.5 to 7.5. Amylase did not aggregate under these conditions, although <10% of parotid secretory protein (PSP) aggregated below pH 6.0. To test aggregation directly in isolated granules, rat parotid secretory granules were permeabilized with 0.1% saponin in the presence or absence of calcium and in the pH range of 5.0 to 8.4. In contrast to the low-pH-dependent retention of amylase in exocrine pancreatic granules, amylase was quantitatively released and most PSP was released from parotid granules under all conditions. Both proteins were completely released upon granule membrane solubilization. Thus neither amylase nor PSP show low-pH- or calcium-induced aggregation under physiological conditions in the exocrine parotid secretory granules.     

SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in Escherichia coli

SecA is an acidic, peripheral membrane protein involved in the translocation of secretory proteins across the cytoplasmic membrane. The direct interaction of SecA with secretory proteins was demonstrated by means of chemical cross-linking with 1-ethyl-3-(3-dimethylaminoprophyl)carbodiimide. OmpF-Lpp, a model secretory protein, carries either an uncleavable or cleavable signal peptide, and mutant secretory proteins derived from uncleavable OmpF-Lpp were used as translocation substrates. The interaction was SecA-specific. None of the control proteins, which are as acidic as SecA, was cross-linked with uncleavable OmpF-Lpp. The interaction was signal peptide-dependent. The interaction was increasingly enhanced as the number of positively charged amino acid residues at the amino-terminal region of the signal peptide was increased, irrespective of the species of amino acid residues donating the charge. Finally, parallelism was observed between the efficiency of interaction and that of translocation among mutant secretory proteins. It is suggested that precursors of secretory proteins interact with SecA to initiate the translocation reaction.     

Exogenous secretory proteins do not inhibit carbamylcholine-stimulated release of pulse-labelled secretory proteins from rat pancreatic tissue slices

Experiments were conducted to test the concept that the results of secretion studies employing pancreatic tissue slices are significantly biased by the distribution of exocrine secretory proteins between the tissue and the incubation medium. The findings demonstrate (1) that the kinetics of release of pulse-labelled secretory proteins from cholinergically-stimulated tissue slices are independent of the concentration of exogenous exocrine secretory proteins and (2) that if there are bidirectional fluxes of secretory proteins across the cell membrane of pancreatic exocrine cells, these fluxes do not account for the fractional release of pulse-labelled secretory proteins.     

Protein secretion in Corynebacterium glutamicum

Corynebacterium glutamicum, a Gram-positive bacterium, has been widely used for the industrial production of amino acids, such as glutamate and lysine, for decades. Due to several characteristics – its ability to secrete properly folded and functional target proteins into culture broth, its low levels of endogenous extracellular proteins and its lack of detectable extracellular hydrolytic enzyme activity – C. glutamicum is also a very favorable host cell for the secretory production of heterologous proteins, important enzymes, and pharmaceutical proteins. The target proteins are secreted into the culture medium, which has attractive advantages over the manufacturing process for inclusion of body expression – the simplified downstream purification process. The secretory process of proteins is complicated and energy consuming. There are two major secretory pathways in C. glutamicum, the Sec pathway and the Tat pathway, both have specific signal peptides that mediate the secretion of the target proteins. In the present review, we critically discuss recent progress in the secretory production of heterologous proteins and examine in depth the mechanisms of the protein translocation process in C. glutamicum. Some successful case studies of actual applications of this secretory expression host are also evaluated. Finally, the existing issues and solutions in using C. glutamicum as a host of secretory proteins are specifically addressed.     

Unconventional secretory routes: direct protein export across the plasma membrane of mammalian cells

The vast majority of extracellular proteins are exported from mammalian cells by the endoplasmic reticulum/Golgi-dependent secretory pathway. For poorly understood reasons, however, a heterogenous group of extracellular proteins has been discovered that does not make use of signal peptide-dependent secretory transport. Both the release mechanisms and the molecular identity of the secretory machines involved have remained elusive. Recent studies now have established a subgroup of unconventional secretory proteins capable of translocating from the cytoplasm directly across the plasma membrane to get access to the exterior of eukaryotic cells. This review aims to focus on a detailed comparison of the subcellular site of membrane translocation of various unconventional secretory proteins such as the proangiogenic molecule fibroblast growth factor-2 (FGF-2) and Leishmania hydrophilic acylated surface protein B (HASP B). A potential link between membrane translocation and quality control as an integral part of unconventional secretory processes is discussed.     

Aggregation chaperones enhance aggregation and storage of secretory proteins in endocrine cells

Calcium-induced aggregation has been proposed to play a role in the sorting and storage of secretory proteins in secretory granules of endocrine cells. The regulation of this process is not known. Hexahistidine epitope tags were used to create aggregation chaperones that enhance the calcium-induced aggregation of secretory granule proteins in vitro. Indeed, 100% recovery of the aggregating target protein was achieved without any modification of the target protein. The aggregation chaperone is not trapped in the aggregates. Co-expression of His(6)-tagged secreted alkaline phosphatase and the regulated secretory protein chromogranin A resulted in an increased chromogranin storage in secretory granules, and stimulated secretion of chromogranin A increased 50%. However, secretion of secreted alkaline phosphatase was not affected by the hexahistidine epitope tag. Thus, calcium-induced aggregation is not a passive process; rather, aggregation and sorting of secretory proteins can be regulated by aggregation chaperones in the secretory pathway of endocrine cells.