Synaptic-like Microvesicles of Neuroendocrine Cells Originate from a Novel Compartment That Is Continuous with the Plasma Membrane and Devoid of Transferrin Receptor

We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18°C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18°C with the membraneimpermeant, cleavable sulfo-NHS-SS–biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37°C originated exclusively from the membranes containing the MesNaaccessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37°C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18° or 37°C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18°C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.     

Rab4 Regulates Formation of Synaptic-like Microvesicles from Early Endosomes in PC12 Cells

Early endosomes in PC12 cells are an important site for the formation of synaptic-like microvesicles and constitutive recycling vesicles. By immunogold electron microscopy, the small GTPase rab4 was localized to early endosomes and numerous small vesicles in the cell periphery and Golgi area of PC12 cells. Overexpression of GTPase-deficient Q67Lrab4 increased the number of early endosome-associated and cytoplasmic vesicles, whereas expression of GDP-bound S22Nrab4 significantly increased the length of early endosomal tubules. In parallel, Q67Lrab4 induced a shift in rab4, VAMP2, and TfR label from early endosomes to peripheral vesicles, whereas S22Nrab4 increased early endosome labeling of all three proteins. These observations were corroborated by early endosome budding assays. Together, our data document a thus far unrecognized role for rab4 in the formation of synaptic-like microvesicles and add to our understanding of the formation of constitutive recycling vesicles from early endosomes.     

膜上tRNA结合蛋白的分离与初步鉴定

用ＴｒｉｔｏｎＸ－１１４分相法分离啤酒酵母的膜总蛋白,经过酵母ｔＲＮＡ分子交联的Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,用０－０．８ｍｏｌ／Ｌ（ＮＨ４０２ＳＯ４梯度缓冲液洗脱ｔＲＮＡ结合的蛋白质。凝胶阻滞电泳实验室鉴定出两种主要的与ｔＲＮＡ分子特异性结合的蛋白质。     

AP‐1A Controls Secretory Granule Biogenesis and Trafficking of Membrane Secretory Granule Proteins

The adaptor protein 1A complex (AP‐1A) transports cargo between the trans‐Golgi network (TGN) and endosomes. In professional secretory cells, AP‐1A also retrieves material from immature secretory granules (SGs). The role of AP‐1A in SG biogenesis was explored using AtT‐20 corticotrope tumor cells expressing reduced levels of the AP‐1A μ1A subunit. A twofold reduction in μ1A resulted in a decrease in TGN cisternae and immature SGs and the appearance of regulated secretory pathway components in non‐condensing SGs. Although basal secretion of endogenous SG proteins was unaffected, secretagogue‐stimulated release was halved. The reduced μ1A levels interfered with the normal trafficking of carboxypeptidase D (CPD) and peptidylglycine α‐amidating monooxygenase‐1 (PAM‐1), integral membrane enzymes that enter immature SGs. The non‐condensing SGs contained POMC products and PAM‐1, but not CPD. Based on metabolic labeling and secretion experiments, the cleavage of newly synthesized PAM‐1 into PHM was unaltered, but PHM basal secretion was increased in sh‐μ1A PAM‐1 cells. Despite lacking a canonical AP‐1A binding motif, yeast two‐hybrid studies demonstrated an interaction between the PAM‐1 cytosolic domain and AP‐1A. Coimmunoprecipitation experiments with PAM‐1 mutants revealed an influence of the luminal domains of PAM‐1 on this interaction. Thus, AP‐1A is crucial for normal SG biogenesis, function and composition.      

Distinct Roles of Myosin Va in Membrane Remodeling and Exocytosis of Secretory Granules

Hormone‐ and neuropeptide‐containing secretory granules (SGs) of neuroendocrine PC12 cells are formed at the trans‐ Golgi network as immature SGs. These intermediates are converted to mature SGs in a complex maturation process, including matrix condensation, processing of cargo proteins and removal of proteins and membrane in clathrin‐coated vesicles. The resulting mature SGs undergo Ca²⁺‐dependent exocytosis upon an appropriate stimulus. We here show that the motor protein myosin Va is implicated in a maturation step of SGs, their binding to F‐actin and their stimulated exocytosis. Interference with myosin Va function blocked the removal of the transmembrane protein furin from maturing SGs without affecting condensation and processing of proteins of the SG lumen. Furthermore, the ATP‐inhibited binding of SGs to F‐actin decreased with progressive maturation and upon interference with myosin Va function. Moreover, the expression of a dominant‐negative myosin Va‐tail or shRNA‐based downregulation of myosin Va interfered with stimulated exocytosis of SGs. In summary, our data suggest an essential function of myosin Va in the membrane remodeling of SGs during maturation and a role in their exocytosis.     

Roles of Myosin Va and Rab3D in Membrane Remodeling of Immature Secretory Granules

Neuroendocrine secretory granules (SGs) are formed at the trans-Golgi network (TGN) as immature intermediates. In PC12 cells, these immature SGs (ISGs) are transported within seconds to the cell cortex, where they move along actin filaments and complete maturation. This maturation process comprises acidification-dependent processing of cargo proteins, condensation of the SG matrix, and removal of membrane and proteins not destined to mature SGs (MSGs) into ISG-derived vesicles (IDVs). We investigated the roles of myosin Va and Rab3 isoforms in the maturation of ISGs in neuroendocrine PC12 cells. The expression of dominant-negative mutants of myosin Va or Rab3D blocked the removal of the endoprotease furin from ISGs. Furthermore, expression of mutant Rab3D, but not of mutant myosin Va, impaired cargo processing of SGs. In conclusion, our data suggest an implication of myosin Va and Rab3D in the maturation of SGs where they participate in overlapping but not identical tasks.     

Manipulation of Disulfide Bonds Differentially Affects the Intracellular Transport, Sorting, and Processing of Neuroendocrine Secretory Proteins

Abstract: To investigate if the prevention of disulfide bond formation affects the intracellular transport, sorting, and processing of a distinct set of neuroendocrine proteins in the regulated secretory pathway, we have treated Xenopus intermediate pituitaries with the thiol-reducing agent dithiothreitol. Pulse-chase incubations in combination with immunoprecipitation analysis were used to monitor the fates of the prohormone proopiomelanocortin (POMC), prohormone convertase PC2 and its helper protein 7B2, as well as secretogranin III. Manipulation of the disulfide bonds in POMC and proPC2 blocked their transport to the trans -Golgi network and strongly inhibited their processing. Reduction of the single disulfide bond in 7B2 did not disturb its transport and cleavage, but caused its missorting to the constitutive secretory pathway. Moreover, the liaison between proPC2 and 7B2 was prevented. Dithiothreitol did not affect transport, sorting, and cleavage of secretogranin III, which lacks disulfide bonds. When the reducing agent was washed away, POMC processing, proPC2 maturation, and the association between proPC2 and 7B2 were reestablished. Collectively, our findings indicate that manipulation of disulfide bonds differentially affects the fates of neuroendocrine proteins during their transit through the secretory pathway.     

Role of Tim50 in the Transfer of Precursor Proteins from the Outer to the Inner Membrane of Mitochondria

Transport of essentially all matrix and a number of inner membrane proteins is governed, entirely or in part, by N-terminal presequences and requires a coordinated action of the translocases of outer and inner mitochondrial membranes (TOM and TIM23 complexes). Here, we have analyzed Tim50, a subunit of the TIM23 complex that is implicated in transfer of precursors from TOM to TIM23. Tim50 is recruited to the TIM23 complex via Tim23 in an interaction that is essentially independent of the rest of the translocase. We find Tim50 in close proximity to the intermembrane space side of the TOM complex where it recognizes both types of TIM23 substrates, those that are to be transported into the matrix and those destined to the inner membrane, suggesting that Tim50 recognizes presequences. This function of Tim50 depends on its association with TIM23. We conclude that the efficient transfer of precursors between TOM and TIM23 complexes requires the concerted action of Tim50 with Tim23.     

HPLC Analysis of Neurophysin Proteins from Neurosecretory Granules

Abstract: Neurosecretory granules were obtained from neurolobes of porcine pituitary glands. From the granules, highly purified neurophysins were prepared by HPLC. According to polyacrylamide gel electrophoresis, isoelectric focusing, N- and C-terminal amino acid residue determination, and amino acid composition, the neurophysins I₁ I₂, and II were identical to the neurophysins obtained from whole posterior lobes. Since degradation could not have occurred, we conclude that neurophysin I₁ and I₂ originated in the neurosecretory granules.     

Immunological Characterization of Secretory Proteins of Chromaffin Granules: Chromogranins A, Chromogranins B, and Enkephalin-Containing Peptides

The soluble proteins of bovine chromaffin granules can be resolved into about 40 proteins by two-dimensional electrophoresis. Use of several antisera enabled us to characterize most of these proteins with the immune replica technique. An antiserum against dopamine beta-hydroxylase reacted with one protein of Mr 75,000. Met-enkephalin antisera labeled eight proteins of Mr 23,000-14,000. A new method was developed to obtain highly purified chromogranin A for immunization. The antiserum reacted with chromogranin A and several smaller proteins of similar pI. This specific antiserum did not react with a second family of hitherto undescribed proteins, which we propose to call chromogranins B. An antiserum against these proteins was raised. It labeled several proteins ranging in Mr from 100,000 to 24,000 and focusing at pH 5.2. Subcellular fractionation established that chromogranins B are specifically localized in chromaffin granules of several species. They are secreted from the adrenal medulla during cholinergic stimulation. We conclude that apart from dopamine beta-hydroxylase chromaffin granules contain three families of immunologically unrelated proteins.     

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 Å. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.     

芽胞杆菌高效分泌表达异源蛋白的研究进展

外泌蛋白易于分离纯化,有利于以较低的生产成本获得大量的目标蛋白,因此分泌表达是一种理想的外源基因的表达方式。芽胞杆菌由于其具有较好的分泌能力,被认为是一种理想的表达外源基因的宿主。到目前为止已有大量来源于不同生物的外源基因在芽胞杆菌中实现了高效分泌表达。但是芽胞杆菌的分泌表达系统仍然存在很多问题,如对某些目的蛋白的分泌量低等,限制了其作为“细胞工厂”生产目标蛋白的应用。综述了芽胞杆菌的外泌系统,分析了芽胞杆菌分泌蛋白过程中的限制因素,并总结了相关的解决方案。     

Mimetic Membrane System to Carry Multiple Antigenic Proteins from Leishmania amazonensis

Liposomes have long been used as models for lipid membranes and for the reconstitution of a single or multiple proteins. Also, liposomes have adjuvant activity in vaccines against several protozoan or bacterial organisms. Thus, the main objective of the present study was to obtain a crude extract of detergent-solubilized proteins of Leishmania amazonensis amastigotes and reconstitute them into liposomes. Neutral and zwiterionic detergents were less efficient than an ionic detergent. In order to obtain efficient solubilization using only sodium dodecyl sulfate (SDS), the effects of detergent and protein concentration and incubation time were studied. The maximum of solubilized proteins was obtained instantaneously using a ratio of 0.5 mg/ml of protein to 0.1% (w/v) detergent at 4°C. Dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS) and cholesterol in a weight ratio of 5:1:4 were used for protein reconstitution into liposomes using the cosolubilization method, yielding 60% of incorporation. The incorporation of multiple parasite proteins results in a vesicular diameter of proteoliposomes of about 140 nm, presenting a final lipid weight ratio for DPPC, DPPS and cholesterol of 1:1:5, with high stability. The detergent-solubilized proteins of L. amazonensis amastigotes present in the proteoliposome, when analyzed by SDS-polyacrylamide gel electrophoresis, include a wide range of parasite-incorporated proteins. BALB/c mice inoculated with these proteoliposomes were able to produce antibodies against the proteins reconstituted in DPPC:DPPS:cholesterol liposomes and were partially resistant to infection with L. amazonensis promastigotes. These results indicate that this system can be used as a possible vaccine against L. amazonensis.     

Adriamycin Binds to the Matrix of Secretory Granules during Mast Cell Exocytosis

The antineoplastic drug adriamycin induces exocytosis in rat peritoneal mast cells followed by a significant uptake of the drug into the secretory granules. The drug is fluorescent, allowing visualization of its accumulation and binding to mast cell granules by fluorescence microscopy. At the same time, the well known inorganic dye ruthenium red was used as a probe because of its great affinity for heparin in the mast cell secretory granules as visualized by bright field microscopy. Competition between adriamycin and ruthenium red for binding to the negatively charged matrix of granules was demonstrated. Biochemical studies were also performed to confirm microscopic observations. Adriamycin may be of interest for studying mast cell secretion; it is not only a strong fluorescent dye for mast cell granules that are in communication with the extracellular space, but it also induces mast cell exocytosis.     

膜蛋白的拓扑学

膜蛋白的拓扑学是研究膜蛋白三维结构的出发点．利用融合蛋白和化学修饰等实验技术已确定了很多膜蛋白的拓扑学．对膜蛋白的转运与插膜的研究确定可能存在两类插膜元件．对已知拓扑学的膜蛋白的统计分析以及蛋白质工程的研究表明存在膜蛋白拓扑学的内正规则．目前已形成预测膜蛋白的拓扑学的比较可靠的策略,这在反向生物学上具有重要意义.但要进行三维结构的预测还有许多路要走．     

Preferential Transfer of Certain Plasma Membrane Proteins onto T and B Cells by Trogocytosis

T and B cells capture antigens via membrane fragments of antigen presenting cells (APC) in a process termed trogocytosis. Whether (and how) a preferential transfer of some APC components occurs during trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM). Only the latter were transferred by trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM''s cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM''s whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRγ found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis.