Essential Role of the Disulfide-bonded Loop of Chromogranin B for Sorting to Secretory Granules Is Revealed by Expression of a Deletion Mutant in the Absence of Endogenous Granin Synthesis

Sorting of regulated secretory proteins in the TGN to immature secretory granules (ISG) is thought to involve at least two steps: their selective aggregation and their interaction with membrane components destined to ISG. Here, we have investigated the sorting of chromogranin B (CgB), a member of the granin family present in the secretory granules of many endocrine cells and neurons. Specifically, we have studied the role of a candidate structural motif implicated in the sorting of CgB, the highly conserved NH₂-terminal disulfide– bonded loop. Sorting to ISG of full-length human CgB and a deletion mutant of human CgB (Δcys-hCgB) lacking the 22–amino acid residues comprising the disulfide-bonded loop was compared in the rat neuroendocrine cell line PC12. Upon transfection, i.e., with ongoing synthesis of endogenous granins, the sorting of the deletion mutant was only slightly impaired compared to full-length CgB. To investigate whether this sorting was due to coaggregation of the deletion mutant with endogenous granins, we expressed human CgB using recombinant vaccinia viruses, under conditions in which the synthesis of endogenous granins in the infected PC12 cells was shut off. In these conditions, Δcys-hCgB, in contrast to full-length hCgB, was no longer sorted to ISG, but exited from the TGN in constitutive secretory vesicles. Coexpression of full-length hCgB together with Δcys-hCgB by double infection, using the respective recombinant vaccinia viruses, rescued the sorting of the deletion mutant to ISG. In conclusion, our data show that (a) the disulfide-bonded loop is essential for sorting of CgB to ISG and (b) the lack of this structural motif can be compensated by coexpression of loop-bearing CgB. Furthermore, comparison of the two expression systems, transfection and vaccinia virus–mediated expression, reveals that analyses under conditions in which host cell secretory protein synthesis is blocked greatly facilitate the identification of sequence motifs required for sorting of regulated secretory proteins to secretory granules.     

Brefeldin A inhibits the formation of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network.

The effects of brefeldin A (BFA) on membrane traffic between the trans-Golgi network (TGN) and the plasma membrane were investigated in intact PC12 cells and in a cell-free system derived from PC12 cells. In intact cells, BFA caused a virtually complete block of constitutive secretion, as indicated by the lack of release from, and accumulation in, the cells of a [35S]sulfate-labeled heparan sulfate proteoglycan (hsPG). Pulse-chase experiments with [35S]sulfate followed by subcellular fractionation showed that this block was due to the inhibition of formation of constitutive secretory vesicles (CSVs) from the TGN. BFA did not block the depolarization-induced release of [35S]sulfate-labeled chromogranin B (CgB) and secretogranin II (SgII) from secretory granules formed prior to the addition of the drug, showing that BFA does not block secretory granule fusion with the plasma membrane. The presence of BFA did, however, prevent the appearance of [35S]sulfate-labeled CgB and SgII in secretory granules, indicating that the drug inhibits the formation of secretory granules from the TGN. Evidence for a direct block of vesicle formation by BFA was obtained using a cell-free system derived from [35S]sulfate-labeled PC12 cells. In this system, low concentrations of BFA (5 micrograms/ml) inhibited the formation of the hsPG-containing CSVs and that of the SgII-containing secretory granules from the TGN to the same extent (50-60%) as, and in a non-additive manner with, the nonhydrolyzable GTP analogue GTP gamma S. Consistent with the inhibitory effects of BFA on vesicle formation from the TGN, BFA treatment of intact PC12 cells led to the hypersialylation of CgB, which presumably was due to the increased residence time of the protein in the TGN. In conclusion, our data are consistent with, and allow the generalization of, the concept that the BFA-induced block of anterograde membrane traffic results from the inhibition of vesicle formation from a donor compartment.     

Reduction of the disulfide bond of chromogranin B (secretogranin I) in the trans-Golgi network causes its missorting to the constitutive secretory pathways.

The role of the single, highly conserved disulfide bond in chromogranin B (secretogranin I) on the sorting of this regulated secretory protein to secretory granules was investigated in the neuroendocrine cell line PC12. Treatment of PC12 cells with dithiothreitol (DTT), a membrane permeable thiol reducing agent known to prevent disulfide bond formation in intact cells, resulted in the secretion of newly synthesized chromogranin B, but only slightly decreased the intracellular storage of newly synthesized secretogranin II, a regulated secretory protein devoid of cysteines. The secretion of newly synthesized chromogranin B in the presence of DTT occurred with similar kinetics to those of a heparan sulfate proteoglycan, a known marker of the constitutive secretory pathway in PC12 cells. Analysis of the various secretory vesicles derived from the trans-Golgi network (TGN) indicated that DTT treatment diverted newly synthesized chromogranin B to constitutive secretory vesicles, whereas the packaging of secretogranin II into immature secretory granules was unaffected by the reducing agent. The chromogranin B molecules diverted to constitutive secretory vesicles, in contrast to those stored in secretory granules, were found to contain free sulfhydryl residues. The effect of DTT on chromogranin B occurred in the TGN rather than in the endoplasmic reticulum. We conclude that the sorting of CgB in the TGN to secretory granules is dependent upon the integrity of its single disulfide bond.     

Chromogranin B (secretogranin I), a neuroendocrine-regulated secretory protein, is sorted to exocrine secretory granules in transgenic mice.

Chromogranin B (CgB, secretogranin I) is a secretory granule matrix protein expressed in a wide variety of endocrine cells and neurons. Here we generated transgenic mice expressing CgB under the control of the human cytomegalovirus promoter. Northern and immunoblot analyses, in situ hybridization and immunocytochemistry revealed that the exocrine pancreas was the tissue with the highest level of ectopic CgB expression. Upon subcellular fractionation of the exocrine pancreas, the distribution of CgB in the various fractions was indistinguishable from that of amylase, an endogenous constituent of zymogen granules. Immunogold electron microscopy of pancreatic acinar cells showed co-localization of CgB with zymogens in Golgi cisternae, condensing vacuoles/immature granules and mature zymogen granules; the ratio of immunoreactivity of CgB to zymogens being highest in condensing vacuoles/immature granules. CgB isolated from zymogen granules of the pancreas of the transgenic mice aggregated in a mildly acidic (pH 5.5) milieu in vitro, suggesting that low pH-induced aggregation contributed to the observed concentration of CgB in condensing vacuoles. Our results show that a neuroendocrine-regulated secretory protein can be sorted to exocrine secretory granules in vivo, and imply that a key feature of CgB sorting in the trans-Golgi network of neuroendocrine cells, i.e. its aggregation-mediated concentration in the course of immature secretory granule formation, also occurs in exocrine cells although secretory protein sorting in these cells is thought to occur largely in the course of secretory granule maturation.     

Interaction of furin in immature secretory granules from neuroendocrine cells with the AP-1 adaptor complex is modulated by casein kinase II phosphorylation.

The composition of secretory granules in neuroendocrine and endocrine cells is determined by two sorting events; the first in the trans-Golgi complex (TGN), the second in the immature secretory granule (ISG). Sorting from the ISG, which may be mediated by the AP-1 type adaptor complex and clathrin-coated vesicles, occurs during ISG maturation. Here we show that furin, a ubiquitously expressed, TGN/endosomal membrane endoprotease, is present in the regulated pathway of neuroendocrine cells where it is found in ISGs. By contrast, TGN38, a membrane protein that is also routed through the TGN/endosomal system does not enter ISGs. Furin, however, is excluded from mature secretory granules, suggesting that the endoprotease is retrieved from the clathrin-coated ISGs. Consistent with this, we show that the furin cytoplasmic domain interacts with AP-1, a component of the TGN/ISG-localized clathrin sorting machinery. Interaction between AP-1 and furin is dependent on phosphorylation of the enzyme's cytoplasmic domain by casein kinase II. Finally, in support of a requirement for the phosphorylation-dependent association of furin with AP-1, expression of furin mutants that mimic either the phosphorylated or unphosphorylated forms of the endoprotease in AtT-20 cells demonstrates that the integrity of the CKII sites is necessary for removal of furin from the regulated pathway.     

Chromogranin B (secretogranin I), a secretory protein of the regulated pathway, is also present in a tightly membrane-associated form in PC12 cells.

Chromogranin B (CgB, also called secretogranin I) is a secretory protein sorted to secretory granules in a wide variety of endocrine cells and neurons. Unexpectedly, after stimulation of regulated secretion in the neuroendocrine cell line PC12, a fraction of the exocytosed CgB was not released into the medium but remained associated with the plasma membrane. The addition of exogenous CgB to unstimulated cells did not result in the appearance of cell surface CgB, suggesting that the presence of cell surface CgB could not be accounted for by adsorption of released CgB to the cell surface. Upon further incubation of stimulated PC12 cells, the surface CgB was internalized by the cells and largely degraded. The surface CgB was not released by exposure to pH 11, yet it partitioned in the aqueous phase upon Triton X-114 phase separation. Subcellular fractionation and differential extraction studies showed that the membrane-associated CgB constituted at least 10% of the total cellular CgB. These observations suggest that (a) the appearance of CgB at the cell surface is due to fusion of secretory granules with the plasma membrane and (b) a fraction of CgB is present in tight association with the secretory granule membrane. We propose a model in which membrane-associated CgB, by virtue of its ability to interact in a homophilic manner with soluble CgB, plays a key role in the sorting and targeting of CgB to the regulated pathway.     

Disruption of disulfide bonds exhibits differential effects on trafficking of regulated secretory proteins

For several secretory proteins, it has beenhypothesized that disulfide-bonded loop structures are required forsorting to secretory granules. To explore this hypothesis, we employeddithiothreitol (DTT) treatment in live pancreatic islets, as well as inPC-12 andGH₄C₁cells. In islets, disulfide reduction in the distal secretory pathwaydid not increase constitutive or constitutive-like secretion ofproinsulin (or insulin). In PC-12 cells, DTT treatment caused adramatic increase in unstimulated secretion of newly synthesizedchromogranin B (CgB), presumably as a consequence of reducing thesingle conserved chromogranin disulfide bond (E. Chanat, U. Weiss, W. B. Huttner, and S. A. Tooze. EMBO J. 12: 2159-2168, 1993). However, inGH₄C₁cells that also synthesize CgB endogenously, DTT treatment reducednewly synthesized prolactin and blocked its export, whereas newlysynthesized CgB was routed normally to secretory granules. Moreover, ontransient expression inGH₄C₁cells, CgA and a CgA mutant lacking the conserved disulfide bond showedcomparable multimeric aggregation properties and targeting to secretorygranules, as measured by stimulated secretion assays. Thus theconformational perturbation of regulated secretory proteins caused bydisulfide disruption leads to consequences in protein trafficking thatare both protein and cell type dependent.

     

Transport via the regulated secretory pathway in semi-intact PC12 cells: role of intra-cisternal calcium and pH in the transport and sorting of secretogranin II

To gain insight into the mechanisms governing protein sorting, we have developed a system that reconstitutes both the formation of immature secretory granules and their fusion with the plasma membrane. Semi- intact PC12 cells were incubated with ATP and cytosol for 15 min to allow immature granules to form, and then in a buffer containing 30 microM [Ca2+]free to induce exocytosis. Transport via the regulated pathway, as assayed by the release of secretogranin II (SgII) labeled in the TGN, was inhibited by depletion of ATP, or by the inclusion of 100 microM GTP gamma S, 50 microM AlF3-5 or 5 micrograms/ml BFA. When added after immature granules had formed, GTP gamma S stimulated rather than inhibited exocytosis. Thus, exocytosis of immature granules in this system resembles the characteristics of fully matured granules. Transport of SgII via the regulated pathway occurred at a fourfold higher efficiency than glycosaminoglycan chains, indicating that SgII is sorted to some extent upon exit from the TGN. Addition of A23187 to release Ca2+ from the TGN had no significant effect on sorting of SgII into immature granules. In contrast, depletion of lumenal calcium inhibited the endoproteolytic cleavage of POMC and proinsulin. These results establish the importance of intra-cisternal Ca2+ in prohormone processing, but raise the question whether lumenal calcium is required for proper sorting of SgII into immature granules. Disruption of organelle pH gradients with an ionophore or a weak base resulted in the inhibition of transport via both the constitutive and the regulated pathways.     

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.     

Secretory granule biogenesis: rafting to the SNARE

Regulated secretion of hormones occurs when a cell receives an external stimulus, triggering the secretory granules to undergo fusion with the plasma membrane and release their content into the extracellular milieu. The formation of a mature secretory granule (MSG) involves a series of discrete and unique events such as protein sorting, formation of immature secretory granules (ISGs), prohormone processing and vesicle fusion. Regulated secretory proteins (RSPs), the proteins stored and secreted from MSGs, contain signals or domains to direct them into the regulated secretory pathway. Recent data on the role of specific domains in RSPs involved in sorting and aggregation suggest that the cell-type-specific composition of RSPs in the trans-Golgi network (TGN) has an important role in determining how the RSPs get into ISGs. The realization that lipid rafts are implicated in sorting RSPs in the TGN and the identification of SNARE molecules represent further major advances in our understanding of how MSGs are formed. At the heart of these findings is the elucidation of molecular mechanisms driving protein--lipid and protein--protein interactions specific for secretory granule biogenesis.     

Recycling of Raft-associated prohormone sorting receptor carboxypeptidase E requires interaction with ARF6

Little is known about the molecular mechanism of recycling of intracellular receptors and lipid raft-associated proteins. Here, we have investigated the recycling pathway and internalization mechanism of a transmembrane, lipid raft-associated intracellular prohormone sorting receptor, carboxypeptidase E (CPE). CPE is found in the trans-Golgi network (TGN) and secretory granules of (neuro)endocrine cells. An extracellular domain of the IL2 receptor alpha-subunit (Tac) fused to the transmembrane domain and cytoplasmic tail of CPE (Tac-CPE25) was used as a marker to track recycling of CPE. We show in (neuro)endocrine cells, that upon stimulated secretory granule exocytosis, raft-associated Tac-CPE25 was rapidly internalized from the plasma membrane in a clathrin-independent manner into early endosomes and then transported through the endocytic recycling compartment to the TGN. A yeast two-hybrid screen and in vitro binding assay identified the CPE cytoplasmic tail sequence S472ETLNF477 as an interactor with active small GTPase ADP-ribosylation factor (ARF) 6, but not ARF1. Expression of a dominant negative, inactive ARF6 mutant blocked this recycling. Mutation of residues S472 or E473 to A in the cytoplasmic tail of CPE obliterated its binding to ARF6, and internalization from the plasma membrane of Tac-CPE25 mutated at S472 or E473 was significantly reduced. Thus, CPE recycles back to the TGN by a novel mechanism requiring ARF6 interaction and activity.     

Localization of metallocarboxypeptidase D in AtT-20 cells. Potential role in prohormone processing.

Carboxypeptidase D (CPD) is a recently discovered metallocarboxypeptidase that is predominantly located in the trans-Golgi network (TGN), and also cycles between the cell surface and the TGN. In the present study, the intracellular distribution of CPD was examined in AtT-20 cells, a mouse anterior pituitary-derived corticotroph. CPD-containing compartments were isolated using antibodies to the CPD cytosolic tail. The immunopurified vesicles contained TGN proteins (TGN38, furin, syntaxin 6) but not lysosomal or plasma membrane proteins. The CPD-containing vesicles also contained neuropeptide-processing enzymes and adrenocorticotropic hormone, a product of proopiomelanocortin proteolysis. Electron microscopic analysis revealed that CPD is present within the TGN and immature secretory granules but is virtually absent from mature granules, suggesting that CPD is actively removed from the regulated pathway during the process of granule maturation. A second major finding of the present study is that a soluble truncated form of CPD is secreted mainly via the constitutive pathway in AtT-20 cells, indicating that the lumenal domain does not contain signals for the sorting of CPD to mature secretory granules. Taken together, these data are consistent with the proposal that CPD participates in the processing of proteins within the TGN and immature secretory vesicles.     

Lipid raft association of carboxypeptidase E is necessary for its function as a regulated secretory pathway sorting receptor

Membrane carboxypeptidase E (CPE) is a sorting receptor for targeting prohormones, such as pro-opiomelanocortin, to the regulated secretory pathway in endocrine cells. Its membrane association is necessary for it to bind a prohormone sorting signal at the trans-Golgi network (TGN) to facilitate targeting. In this study, we examined the lipid interaction of CPE in bovine pituitary secretory granule membranes, which are derived from the TGN. We show that CPE is associated with detergent-resistant lipid domains, or rafts, within secretory granule membranes. Lipid analysis revealed that these rafts are enriched in glycosphingolipids and cholesterol. Pulse-chase and subcellular fractionation experiments in AtT-20 cells show that the association of CPE with membrane rafts occurred only after it reached the Golgi. Cholesterol depletion resulted in dissociation of CPE from secretory granule membranes and decreased the binding of prohormones to membranes. In vivo cholesterol depletion using lovastatin resulted in the lack of sorting of CPE and its cargo to the regulated secretory pathway. We propose that the sorting receptor function of CPE necessitates its interaction with glycosphingolipid-cholesterol rafts at the TGN, thereby anchoring it in position to bind to its prohormone cargo.     

Cofilin-mediated sorting and export of specific cargo from the Golgi apparatus in yeast

The mechanism of cargo sorting at the trans-Golgi network (TGN) for secretion is poorly understood. We previously reported the involvement of the actin-severing protein cofilin and the Ca(2+) ATPase secretory pathway calcium ATPase 1 (SPCA1) in the sorting of soluble secretory cargo at the TGN in mammalian cells. Now we report that cofilin in yeast is required for export of selective secretory cargo at the late Golgi membranes. In cofilin mutant (cof1-8) cells, the cell wall protein Bgl2 was secreted at a reduced rate and retained in a late Golgi compartment, whereas the plasma membrane H(+) ATPase Pma1, which is transported in the same class of carriers, reached the cell surface. In addition, sorting of carboxypeptidase Y (CPY) to the vacuole was delayed, and CPY was secreted from cof1-8 cells. Loss of the yeast orthologue of SPCA1 (Pmr1) exhibited similar sorting defects and displayed synthetic sickness with cof1-8. In addition, overexpression of PMR1 restored Bgl2 secretion in cof1-8 cells. These findings highlight the conserved role of cofilin and SPCA1/Pmr1 in sorting of the soluble secretory proteins at the TGN/late Golgi membranes in eukaryotes.     

Recruitment of arfaptins to the trans‐Golgi network by PI(4)P and their involvement in cargo export

The BAR (Bin/Amphiphysin/Rvs) domain proteins arfaptin1 and arfaptin2 are localized to the trans‐Golgi network (TGN) and, by virtue of their ability to sense and/or generate membrane curvature, could play an important role in the biogenesis of transport carriers. We report that arfaptins contain an amphipathic helix (AH) preceding the BAR domain, which is essential for their binding to phosphatidylinositol 4‐phosphate (PI(4)P)‐containing liposomes and the TGN of mammalian cells. The binding of arfaptin1, but not arfaptin2, to PI(4)P is regulated by protein kinase D (PKD) mediated phosphorylation at Ser100 within the AH. We also found that only arfaptin1 is required for the PKD‐dependent trafficking of chromogranin A by the regulated secretory pathway. Altogether, these findings reveal the importance of PI(4)P and PKD in the recruitment of arfaptins at the TGN and their requirement in the events leading to the biogenesis of secretory storage granules.     

Immunocytochemical localization of secretogranin III in the anterior lobe of male rat pituitary glands.

Secretogranin III (SgIII) is one of the acidic secretory proteins, designated as granins, which are specifically expressed in neuronal and endocrine cells. To clarify its precise distribution in the anterior lobe of the rat pituitary gland, we raised a polyclonal antiserum against rat SgIII for immunocytochemical analyses. By immunohistochemistry using semithin sections, positive signals for SgIII were detected intensely in mammotropes and thyrotropes, moderately in gonadotropes and corticotropes, but not in somatotropes. The distribution pattern of SgIII in the pituitary gland was similar to that of chromogranin B (CgB), also of the granin protein family, suggesting that the expressions of these two granins are regulated by common mechanisms. The localization of SgIII in endocrine cells was confirmed by immunoelectron microscopy. In particular, secretory granules of mammotropes and thyrotropes were densely and preferentially co-labeled for SgIII and CgB in their periphery. Moreover, positive signals for SgIII were occasionally found in cells containing both prolactin and TSH in secretory granules. These lines of evidence suggest that SgIII and CgB are closely associated with the secretory granule membrane and that this membrane association might contribute to gathering and anchoring of other soluble constituents to the secretory granule membrane.     

Visualization of the post-Golgi trafficking of multiphoton photoactivated transferrin receptors

Newly synthesized membrane proteins are sorted in the trans-Golgi network (TGN) on the basis of sorting signals carried in their cytoplasmic domains and delivered to their final destinations in the secretory and endocytic pathways. Although previous studies have suggested the involvement of early endosomes in the biosynthetic pathway of transmembrane proteins, the precise trafficking routes followed by the newly synthesized plasma membrane proteins, such as transferrin receptors (TfRs), after exit from the TGN remain unclear. In this report, first, we demonstrated the advantages of photoactivating PA-GFP, a variant of the Aequorea victoria green fluorescent protein (GFP), with multiphoton laser light rather than single-photon laser light, in terms of photoactivation efficiency and spatial resolution. We then applied the multiphoton photoactivation technique to selectively photoactivate the TfR tagged with PA-GFP (PA-GFP-TfR) at the TGN, and monitored the movement of the photoactivated PA-GFP-TfR in live cells. We observed that the PA-GFP-TfR photoactivated at the TGN are transported to the Tfn(+)EEA1(+) endosomal compartments after exiting the TGN. These data support the notion that early endosomes can serve as a sorting station for not only internalized plasma membrane proteins in the endocytic pathway but also newly synthesized membrane proteins in the post-Golgi secretory pathway.     

Actin remodeling by ADF/cofilin is required for cargo sorting at the trans-Golgi network

Knockdown of the actin-severing protein actin-depolymerizing factor (ADF)/cofilin inhibited export of an exogenously expressed soluble secretory protein from Golgi membranes in Drosophila melanogaster and mammalian tissue culture cells. A stable isotope labeling by amino acids in cell culture mass spectrometry–based protein profiling revealed that a large number of endogenous secretory proteins in mammalian cells were not secreted upon ADF/cofilin knockdown. Although many secretory proteins were retained, a Golgi-resident protein and a lysosomal hydrolase were aberrantly secreted upon ADF/cofilin knockdown. Overall, our findings indicate that inactivation of ADF/cofilin perturbed the sorting of a subset of both soluble and integral membrane proteins at the trans-Golgi network (TGN). We suggest that ADF/cofilin-dependent actin trimming generates a sorting domain at the TGN, which filters secretory cargo for export, and that uncontrolled growth of this domain causes missorting of proteins. This type of actin-dependent compartmentalization and filtering of secretory cargo at the TGN by ADF/cofilin could explain sorting of proteins that are destined to the cell surface.     

Chromogranin B: isolation from pheochromocytoma, N-terminal sequence, tissue distribution and secretory vesicle processing

The chromogranins/secretogranins are a family of neuroendocrine vesicle secretory proteins. Immunohistology and immunoblotting have suggested that a major soluble protein in human chromaffin granules may be chromogranin B (CgB). We purified from pheochromocytoma chromaffin granules an SDS-PAGE 110-120 kDa protein whose N-terminal sequence matched that previously deduced from a human CgB cDNA. An antibody directed against a synthetic human CgB N-terminal region specifically recognized the CgB N-terminus, though not the chromogranin A (CgA) N-terminus or the CgB C-terminus on immunoblots. An antiserum directed against CgB's C-terminus also visualized CgB but not CgA. By immunoblotting, CgB was a quantitatively major protein in human pheochromocytoma chromaffin granules, but a relatively minor in normal bovine adrenal medullary chromaffin granules. In a variety of normal bovine neuroendocrine tissues, the relative abundance of CgB immunoreactivity on immunoblots was: adrenal medulla greater than anterior pituitary greater than pancreas greater than small intestine, hypothalamus. Immunoblotting of neuroendocrine tissues (or their hormone storage vesicle cores) with both anti N-terminal and anti C-terminal CgB antisera suggested bidirectional cleavage or processing of CgB; in the anterior pituitary, a unique 40 kDa C-terminal fragment was observed. Bidirectional CgB cleavage was also suggested on immunoblots of chromaffin tissue from three species (human, bovine, rat). C-terminal processing of CgB was also confirmed by amino acid sequencing of SDS-PAGE-separated, polyvinylidene difluoride membrane-immobilized CgB fragments from pheochromocytoma chromaffin granules. Whether such fragments possess biological activity remains to be investigated.     

Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery.