Post-Golgi protein traffic in the plant secretory pathway

The Golgi apparatus in plants is organized as a multitude of individual stacks that are motile in the cytoplasm and in close association with the endoplasmic reticulum (ER) (Boevink et al. in Plant J 15:441–447, 1998). These stacks operate as a sorting centre for cargo molecules, providing modification and redirection to other organelles as appropriate. In the post-Golgi direction, these include vacuole and plasma membrane, and specialized transport routes to each are required to prevent mislocalization. Recent evidence in plant cells points to the existence of post-Golgi organelles that function as intermediate stations for efficient protein traffic, as well as to the influence of small GTPases such as Rabs and ARFs on post-Golgi trafficking. This review focuses on the latest findings on post-Golgi trafficking routes and on the involvement of GTPases and their effectors on the trafficking of proteins in the plant secretory pathway. Sally L. Hanton and Loren A. Matheson have contributed equally to this work.     

Astrocytes as secretory cells of the central nervous system: idiosyncrasies of vesicular secretion

Astrocytes are housekeepers of the central nervous system (CNS) and are important for CNS development, homeostasis and defence. They communicate with neurones and other glial cells through the release of signalling molecules. Astrocytes secrete a wide array of classic neurotransmitters, neuromodulators and hormones, as well as metabolic, trophic and plastic factors, all of which contribute to the gliocrine system. The release of neuroactive substances from astrocytes occurs through several distinct pathways that include diffusion through plasmalemmal channels, translocation by multiple transporters and regulated exocytosis. As in other eukaryotic cells, exocytotic secretion from astrocytes involves divergent secretory organelles (synaptic‐like microvesicles, dense‐core vesicles, lysosomes, exosomes and ectosomes), which differ in size, origin, cargo, membrane composition, dynamics and functions. In this review, we summarize the features and functions of secretory organelles in astrocytes. We focus on the biogenesis and trafficking of secretory organelles and on the regulation of the exocytotic secretory system in the context of healthy and diseased astrocytes.     

Rab18 inhibits secretory activity in neuroendocrine cells by interacting with secretory granules

Rab proteins comprise a complex family of small GTPases involved in the regulation of intracellular membrane trafficking and reorganization. In this study, we identified Rab18 as a new inhibitory player of the secretory pathway in neuroendocrine cells. In adrenal chromaffin PC12 cells and pituitary AtT20 cells, Rab18 is located at the cytosol but associates with a subpopulation of secretory granules after stimulation of the regulated secretory pathway, strongly suggesting that induction of secretion provokes Rab18 activation and recruitment to these organelles. In support of this, a dominant-inactive Rab18 mutant was found to distribute diffusely in the cytosol, whereas a dominant-active Rab18 mutant was predominantly associated to secretory granules. Furthermore, interaction of Rab18 with secretory granules was associated to an inhibition in the secretory activity of PC12 and AtT20 cells in response to stimulatory challenges. Association of Rab18 with secretory granules was also observed by immunoelectron microscopy in normal, non-tumoral endocrine cells (pituitary melanotropes), wherein Rab18 protein content is inversely correlated to the level of secretory activity of cells. Taken together, these findings suggest that, in neuroendocrine cells, Rab18 acts as a negative regulator of secretory activity, likely by impairing secretory granule transport.     

Tridimensional analysis of the formation of secretory vesicles in the Golgi apparatus of absorptive columnar cells of the mouse colon

The tridimensional structure of the Golgi apparatus has been studied in the absorptive cells of the mouse colon by means of reduced osmium postfixation and phosphatase cytochemistry. In thick sections of tissue impregnated with osmium tetroxide or treated with a technique to demonstrate TPPase activity, the Golgi formed a continuous ribbon-like structure capping the upper pole of the nucleus. Along the longitudinal axis of this ribbon, compact zones made up of superposed flattened saccules alternated with less compact zones which consisted of highly perforated saccules or bridging anastomosed tubules. In the cis-trans axis, the following elements were observed: (1) a cis element consisting of a continuous osmiophilic tubular network; (2) two or three subjacent elements selectively perforated by wells; (3) a trans compartment made up of two or three TPPase-reactive sacculotubular elements, some showing a "peeling-off" configuration. In some regions, the first flattened saccule of this trans compartment displayed discrete ovoid dilatations, located in compact zones and containing a dense granulofibrillar material; in the subjacent elements this material was seen concentrated in nodular swellings, at the intersection of the meshes of anastomosed membranous tubules. 100-300 nm vesicles containing a similar dense granulofilamentous material were observed in the trans Golgi zone and interspersed in the supranuclear cytoplasm between the Golgi zone and the apical surface of the cell. Smaller vesicles 80-100 nm in diameter containing a fine dusty material were also seen in proximity. These morphological observations suggested that at least two kinds of material were segregated in the saccules of the trans compartment and packaged in vesicles of two class sizes that detached from the Golgi stack on its trans aspect.     

Intermediate organelles of the plant secretory pathway: identity and function

The secretory pathway of eukaryotic cells comprises a network of organelles that connects three large membranes, the plasma membrane, the vacuole and the endoplasmic reticulum. The Golgi apparatus and the various post-Golgi organelles that control vacuolar sorting, secretion and endocytosis can be regarded as intermediate organelles of the endocytic and biosynthetic routes. Many processes in the secretory pathway have evolved differently in plants and cannot be studied using yeast or mammalian cells as models. The best characterized organelles are the Golgi apparatus and the prevacuolar compartment, but recent work has shed light on the role of the trans Golgi network, which has to be regarded as a separate organelle in plants. In this study, we wish to highlight recent findings regarding the late secretory pathway and its crosstalk with the early secretory pathway as well as the endocytic route in plants. Recently published findings and suggested models are discussed within the context of known features of the equivalent pathway in other eukaryotes.     

Generic sorting of raft lipids into secretory vesicles in yeast

Previous work has showed that ergosterol and sphingolipids become sorted to secretory vesicles immunoisolated using a chimeric, artificial raft membrane protein as bait. In this study, we have extended this analysis to three populations of secretory vesicles isolated using natural yeast plasma membrane (PM) proteins: Pma1p, Mid2p and Gap1*p as baits. We compared the lipidomes of the immunoisolated vesicles with each other and with the lipidomes of the donor compartment, the trans-Golgi network, and the acceptor compartment, the PM, using a quantitative mass spectrometry approach that provided a complete lipid overview of the yeast late secretory pathway. We could show that vesicles captured with different baits carry the same cargo and have almost identical lipid compositions; being highly enriched in ergosterol and sphingolipids. This finding indicates that lipid raft sorting is a generic feature of vesicles carrying PM cargo and suggests a common lipid-based mechanism for their formation.     

Discovery and progress in our understanding of the regulated secretory pathway in neuroendocrine cells

In this review we start with a historical perspective beginning with the early morphological work done almost 50 years ago. The importance of these pioneering studies is underscored by our brief summary of the key questions addressed by subsequent research into the mechanism of secretion. We then highlight important advances in our understanding of the formation and maturation of neuroendocrine secretory granules, first using in vitro reconstitution systems, then most recently biochemical approaches, and finally genetic manipulations in vitro and in vivo. This work was supported by Fondation pour la Reserche Medicale (FRM 20051105487) and Cancer Research UK.     

Autophagosome formation: Where the secretory and autophagy pathways meet

The upregulation of autophagosome formation in response to nutrient deprivation requires significant intracellular membrane rearrangements that are poorly understood. Recent findings have implicated COPII-coated vesicles, well known as ER-Golgi cargo transport carriers, as key players in macroautophagy. The role of COPII vesicles in macroautophagy and how they interact with autophagy-related (Atg) proteins was unknown. In our recent report, we show that during nutrient deprivation, phosphorylation of the membrane-distal surface of the COPII coat subunit Sec24 facilitates the interaction of Sec24 with the Atg machinery (specifically, Atg9) to regulate the abundance of autophagosomes during starvation. Phosphorylation of Sec24 is specifically required for macroautophagy, but not ER-Golgi transport. These findings begin to unravel the unique function of COPII vesicles during starvation-induced macroautophagy.     

Non-invasive topology analysis of membrane proteins in the secretory pathway

We present a novel method to experimentally visualize in vivo the topology of transmembrane proteins residing in the endoplasmic reticulum (ER) membrane or passing through the secretory pathway on their way to their final destination. This approach, so-called redox-based topology analysis (ReTA), is based on fusion of transmembrane proteins with redox-sensitive GFP (roGFP) and ratiometric imaging. The ratio images provide direct information on the orientation of roGFP relative to the membrane as the roGFP fluorescence alters with changes in the glutathione redox potential across the ER membrane. As proof of concept, we produced binary read-outs using oxidized roGFP inside the ER lumen and reduced roGFP on the cytosolic side of the membrane for both N- and C-terminal fusions of single and multi-spanning membrane proteins. Further, successive deletion of hydrophobic domains from the C-terminus of the K/HDEL receptor ERD2 resulted in alternating localization of roGFP and a topology model for At ERD2 with six transmembrane domains.     

Secretive bacterial pathogens and the secretory pathway

Eukaryotic cells possess two extensive endomembrane systems, each consisting of several sub-compartments connected by vesicular trafficking. One of these systems, the endocytic pathway, serves incoming traffic, and the other system, the secretory pathway (SP), is responsible for surface-bound traffic of intracellularly formed vesicles. Compartments derived of either system can be colonized by intracellular pathogens. In this review, we discuss the interactions between the SP and prominent intracellular bacterial pathogens of the genera Legionella, Brucella, Chlamydia and Salmonella. We emphasize secreted bacterial effector proteins, which directly manipulate host components of this pathway.     

Insights into the mechanism of vascular endothelial cells on bone biology

In the skeletal system, blood vessels not only function as a conduit system for transporting gases, nutrients, metabolic waste, or cells but also provide multifunctional signal molecules regulating bone development, regeneration, and remodeling. Endothelial cells (ECs) in bone tissues, unlike in other organ tissues, are in direct contact with the pericytes of blood vessels, resulting in a closer connection with peripheral connective tissues. Close-contact ECs contribute to osteogenesis and osteoclastogenesis by secreting various cytokines in the paracrine or juxtacrine pathways. An increasing number of studies have revealed that extracellular vesicles (EVs) derived from ECs can directly regulate maturation process of osteoblasts and osteoclasts. The different pathways focus on targets at different distances, forming the basis of the intimate spatial and temporal link between bone tissue and blood vessels. Here, we provide a systematic review to elaborate on the function of ECs in bone biology and its underlying mechanisms based on three aspects: paracrine, EVs, and juxtacrine. This review proposes the possibility of a therapeutic strategy targeting blood vessels, as an adjuvant treatment for bone disorders.     

Unintended molecular interactions in transgenic plants expressing clinically useful proteins: The case of bovine aprotinin traveling the potato leaf cell secretory pathway

We assessed the impact of subcellular targeting on the heterologous expression of a clinically useful protease inhibitor, bovine aprotinin, in leaves of potato, Solanum tuberosum. Transgenic potato lines targeting aprotinin to the cytosol, the ER or the apoplast were first generated, and then assessed for their ability to accumulate the recombinant protein. On‐chip detection and quantitation of aprotinin variants by SELDI TOF MS showed the inhibitor to be absent in the cytosol, but present under different forms in the ER and the apoplast. No visible phenotypic effects of aprotinin were observed for the transgenic lines, but aprotinin retention in the ER was associated with a significant decrease of leaf soluble protein content. A 2‐D gel assessment of control and transgenic lines revealed a possible link between this altered protein content and the down‐regulation of proteins implicated in protein synthesis and maturation. These observations, supported by complementary 2‐DE analyses with potato lines targeting aprotinin to the apoplast, suggest an aprotinin‐mediated feedback in planta negatively altering protein anabolism. From a practical viewpoint, these data illustrate the importance of taking into account not only the characteristics of recombinant proteins expressed in heterologous environments, but also their possible effects on protein accumulation in the host plant factory.     

The Arg617-Arg618 cleavage site in the C-terminal domain of PC1 plays a major role in the processing and targeting of the enzyme within the regulated secretory pathway

The C-terminal domain of the prohormone convertase PC1 is involved in targeting of the enzyme to secretory granules in neuroendocrine cells and is subsequently processed in this compartment at an Arg617-Arg618 site. Three other dibasics are found in the C-terminal domain of mouse PC1. Here, we examined the role of the four dibasics in targeting PC1 to secretory granules. All 15 possible combinations of dibasic mutations were performed. Wild-type (WT) and mutant PC1 were stably expressed in neuroendocrine PC12 cells that lacked endogenous PC1. Processing, secretion and intracellular localization of PC1 and its mutants were analyzed. Leaving intact Arg617-Arg618 and mutating any combination of the three other dibasics yielded proteins that were stored and processed in secretory granules, similarly to WT PC1. Mutating Arg617-Arg618 alone or with any one of the three remaining dibasics generated proteins that were efficiently stored in secretory granules but were not processed further. Mutating Arg617-Arg618 with more than one of the remaining dibasics produced proteins that reached the TGN but were not stored in secretory granules and exited the cells through the constitutive secretory pathway. These data demonstrate that the Arg617-Arg618 plays a prominent role in targeting PC1 to secretory granules.     

GFP-based evaluation system of recombinant expression through the secretory pathway in insect cells and its application to the extracellular domains of class C GPCRs

Applications of the GFP-fusion technique have greatly facilitated evaluations of the amounts and qualities of sample proteins used for structural analyses. In this study, we applied the GFP-based sample evaluation to secreted protein expression by insect cells. We verified that a GFP variant, GFPuv, retains proper folding and monodispersity within all expression spaces in Sf9 cells, such as the cytosol, organelles, and even the extracellular space after secretion, and thus can serve as a proper folding reporter for recombinant proteins. We then applied the GFPuv-based system to the extracellular domains of class C G-protein coupled receptors (GPCRs) and examined their localization, folding, and oligomerization upon insect cell expression. The extracellular domain of metabotropic glutamate receptor 1 (mGluR1) exhibited good secreted expression by Sf9 cells, and the secreted proteins formed dimer with a monodisperse hydrodynamic state favorable for crystallization, consistent with the results from previous successful structural analyses. In contrast, the extracellular domains of sweet/umami taste receptors (T1R) almost completely remained in the cell. Notably, the T1R and mGluR1 subfractions that remained in the cellular space showed polydisperse hydrodynamic states with large aggregated fractions, without forming dimers. These results indicated that the proper folding and oligomerization of the extracellular domains of the class C GPCR are achieved through the secretory pathway.     

Glyceraldehyde-3-phosphate dehydrogenase is required for vesicular transport in the early secretory pathway

Protein transport in the early secretory pathway requires Rab2 GTPase. This protein promotes the recruitment of soluble components that participate in protein sorting and recycling from pre-Golgi intermediates (vesicular tubular clusters (VTCs)). We previously reported that a constitutively activated form of Rab2 (Q65L) as well as Rab2 wild type promoted vesicle formation from VTCs. These vesicles contained Rab2, beta-COP, p53/gp58, and protein kinase Ciota/lambda but lacked anterograde-directed cargo. To identify other candidate Rab2 effectors, the polypeptide composition of the vesicles was further analyzed. We found that vesicles released in response to Rab2 also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To study the relationship of this enzyme to Rab2 function, we performed a quantitative binding assay to measure recruitment of GAPDH to membrane when incubated with Rab2. Rab2-treated microsomes showed a 5-10-fold increase in the level of membrane-associated GAPDH. We generated an affinity-purified anti-GAPDH polyclonal to study the biochemical role of GAPDH in the early secretory pathway. The antibody arrests transport of a reporter molecule in an assay that reconstitutes ER to Golgi traffic. Furthermore, the affinity-purified antibody blocked the ability of Rab2 to recruit GAPDH to membrane. However, the antibody did not interfere with Rab2 stimulated vesicle release. These data suggest that GAPDH is required for ER to Golgi transport. We propose that membranes incubated with anti-GAPDH and Rab2 form "dead end" vesicles that are unable to transport and fuse with the acceptor compartment.     

Functional and immunocytochemical evidence for the expression and localization of the secretory pathway Ca2+-ATPase isoform 1 (SPCA1) in cerebellum relative to other Ca2+ pumps

Membrane fractions of pig cerebellum show Ca2+-ATPase activity and Ca2+ transport due to the presence of the secretory pathway Ca2+-ATPase (SPCA). The SPCA1 isoform shows a wide distribution in the neurons of pig cerebellum, where it is found in the Golgi complex of the soma of Purkinje, stellate, basket and granule cells, and also in more distal components of the secretory pathway associated with a synaptic localization such as in cerebellar glomeruli. The SPCA1 may be involved in loading the Golgi complex and the secretory vesicles of these specific neuronal cell types with Ca2+ and also Mn2+. This study of the cellular and subcellular localization of SPCA1 pumps relative to the sarco(endo) plasmic reticulum Ca2+-ATPase and plasma membrane Ca2+-ATPase pumps hints to a possible specific role of SPCA1 in controlling the luminal secretory pathway Ca2+ (or Mn2+) levels as well as the local cytosolic Ca2+ levels. In addition, it helps to specify the zones that are most vulnerable to Ca2+ and/or Mn2+ dyshomeostasis, a condition that is held responsible of an increasing number of neurological disorders.     

Ultrastructural evidence for the presence of secretory cells in the pineal parenchyma ofHeteropneustes fossilis

There is an extensive literature dealing with the study of indoles, especially serotonin and melatonin, but with considerably less emphasis on the cells and cell types involved in the synthetic process. In the present electron microscopical investigation of the pineal end vesicle ofHeteropneustes fossilis, pinealocytes have been revealed in the pineal parenchyma characterized with extensive synthetic apparatus viz., rough endoplasmic reticulum, free ribosomes, lipid droplets, mitochondria and Golgi bodies. Two sub-populations of the pinealocytes are easily distinguishable on the basis of electron opacity and the preponderance of one or other morphological profile: light cells and dark cells. Light cells represent the active phase of secretion while dark cells represent the storage and release phase of secretion. A neuroendocrine role for the pineal body inHeteropneustes fossilis is suggested which may be significant in view of the nocturnal habit of the fish. Dedicated to my father, Prof. C B L Srivastava     

带有报告基因的板栗疫病菌寄生隐赤壳分泌表达突变体库的构建及分泌缺陷突变体的筛选

丝状真菌分泌蛋白与其致病性密切相关,目前对于病原真菌的蛋白胞外分泌途径及其调控机制的报道不多.为建立一个方便高效的真菌分泌蛋白调控途径的遗传研究体系,本研究以植物病原丝状真菌——板栗疫病菌寄生隐赤壳Cryphonectria parasitica为对象,选取分泌表达量最高的两个分泌蛋白的信号肽SP1和SP2,分别构建带...     

Complex epidermal organs of Phascolion (Sipuncula): insights into the evolution of bimodal secretory cells in annelids

The epidermal organs of an undescribed Phascolion species from the Balearic Islands were investigated using SEM, TEM, LM, CLSM and μCT methods. We found axial receptor cells confirming the previously assumed sensory function of epidermal organs. Our analyses also revealed six types of secretory cells. Some secretory cells types are capable of secreting filamentous and amorphous secretion in two different ways simultaneously (bimodal secretion). The high diversity of cell types, the complex pattern of acinar units, and the absence of a common gland pore make epidermal organs of Phascolion unique amongst sipunculans (Phascolion type). Our reconstruction of the evolution of the epidermal organs of Sipuncula revealed that Phascolion‐type epidermal organs may have derived from either Golfingia‐, Sipunculus‐ or Phascolosoma‐type epidermal organs. The oldest known sipunculans were Golfingia‐like and had epidermal organs, which might resemble the architecture of the Golfingia‐type epidermal organs in extant taxa. Thus, it can be hypothesized that bimodal secretory cells (e.g. basophilic secretory cells) were part of the sipunculan ground pattern. Moreover, bimodal secretory cells of Phascolion look strikingly similar to those found in various annelid glands and thus might even be part of the ground pattern of stem species of Sipuncula + Pleistoannelida.