The Rice α-Amylase Glycoprotein Is Targeted from the Golgi Apparatus through the Secretory Pathway to the Plastids

The well-characterized secretory glycoprotein, rice (Oryza sativa) α-amylase isoform I-1 (AmyI-1), was localized within the plastids and proved to be involved in the degradation of starch granules in the organelles of rice cells. In addition, a large portion of transiently expressed AmyI-1 fused to green fluorescent protein (AmyI-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa) epidermal cells. The plastid targeting of AmyI-1 was inhibited by both dominant-negative and constitutively active mutants of Arabidopsis thaliana ARF1 and Arabidopsis SAR1, which arrest endoplasmic reticulum-to-Golgi traffic. In cells expressing fluorescent trans-Golgi and plastid markers, these fluorescent markers frequently colocalized when coexpressed with AmyI-1. Three-dimensional time-lapse imaging and electron microscopy of high-pressure frozen/freeze-substituted cells demonstrated that contact of the Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids occur within the cells. The transient expression of a series of C-terminal-truncated AmyI-1-GFP fusion proteins in the onion cell system showed that the region from Trp-301 to Gln-369 is necessary for plastid targeting of AmyI-1. Furthermore, the results obtained by site-directed mutations of Trp-302 and Gly-354, located on the surface and on opposite sides of the AmyI-1 protein, suggest that multiple surface regions are necessary for plastid targeting. Thus, Golgi-to-plastid traffic appears to be involved in the transport of glycoproteins to plastids and plastid targeting seems to be accomplished in a sorting signal–dependent manner.     

Is cytochrome P-450 transported from the endoplasmic reticulum to the Golgi apparatus in rat hepatocytes?

The Golgi apparatus mediates intracellular transport of not only secretory and lysosomal proteins but also membrane proteins. As a typical marker membrane protein for endoplasmic reticulum (ER) of rat hepatocytes, we have selected phenobarbital (PB)-inducible cytochrome P- 450 (P-450[PB]) and investigated whether P-450(PB) is transported to the Golgi apparatus or not by combining biochemical and quantitative ferritin immunoelectron microscopic techniques. We found that P-450(PB) was not detectable on the membrane of Golgi cisternae either when P-450 was maximally induced by phenobarbital treatment or when P-450 content in the microsomes rapidly decreased after cessation of the treatment. The P-450 detected biochemically in the Golgi subcellular fraction can be explained by the contamination of the microsomal vesicles derived from fragmented ER membranes to the Golgi fraction. We conclude that when the transfer vesicles are formed by budding on the transitional elements of ER, P-450 is completely excluded from such regions and is not transported to the Golgi apparatus, and only the membrane proteins destined for the Golgi apparatus, plasma membranes, or lysosomes are selectively collected and transported.     

Glucolipotoxicity Impairs Ceramide Flow from the Endoplasmic Reticulum to the Golgi Apparatus in INS-1 β-Cells

Accumulating evidence suggests that glucolipotoxicity, arising from the combined actions of elevated glucose and free fatty acid levels, acts as a key pathogenic component in type II diabetes, contributing to β-cell dysfunction and death. Endoplasmic reticulum (ER) stress is among the molecular pathways and regulators involved in these negative effects, and ceramide accumulation due to glucolipotoxicity can be associated with the induction of ER stress. Increased levels of ceramide in ER may be due to enhanced ceramide biosynthesis and/or decreased ceramide utilization. Here, we studied the effect of glucolipotoxic conditions on ceramide traffic in INS-1 cells in order to gain insights into the molecular mechanism(s) of glucolipotoxicity. We showed that glucolipotoxicity inhibited ceramide utilization for complex sphingolipid biosynthesis, thereby reducing the flow of ceramide from the ER to Golgi. Glucolipotoxicity impaired both vesicular- and CERT-mediated ceramide transport through (1) the decreasing of phospho-Akt levels which in turn possibly inhibits vesicular traffic, and (2) the reducing of the amount of active CERT mainly due to a lower protein levels and increased protein phosphorylation to prevent its localization to the Golgi. In conclusion, our findings provide evidence that glucolipotoxicity-induced ceramide overload in the ER, arising from a defect in ceramide trafficking may be a mechanism that contributes to dysfunction and/or death of β-cells exposed to glucolipotoxicity.     

PDMP Blocks Brefeldin A–induced Retrograde Membrane Transport from Golgi to ER: Evidence for Involvement of Calcium Homeostasis and Dissociation from Sphingolipid Metabolism

In this study, we show that an inhibitor of sphingolipid biosynthesis, d,l-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when BFA was allowed to exert its effect before PDMP addition, PDMP could not “rescue” the Golgi compartment.Evidence is presented showing that this action of PDMP is indirect, which means that the direct target is not sphingolipid metabolism at the Golgi apparatus. A fluorescent analogue of PDMP, 6-(N-[7-nitro-2,1,3-benzoxadiazol-4-yl]amino)hexanoyl-PDMP (C₆-NBD-PDMP), did not localize in the Golgi apparatus. Moreover, the effect of PDMP on membrane flow did not correlate with impaired C₆-NBD-sphingomyelin biosynthesis and was not mimicked by exogenous C₆-ceramide addition or counteracted by exogenous C₆-glucosylceramide addition. On the other hand, the PDMP effect was mimicked by the multidrug resistance protein inhibitor MK571.The effect of PDMP on membrane transport correlated with modulation of calcium homeostasis, which occurred in a similar concentration range. PDMP released calcium from at least two independent calcium stores and blocked calcium influx induced by either extracellular ATP or thapsigargin. Thus, the biological effects of PDMP revealed a relation between three important physiological processes of multidrug resistance, calcium homeostasis, and membrane flow in the ER/ Golgi system.     

Identification of sorting motifs of AtβFruct4 for trafficking from the ER to the vacuole through the Golgi and PVC

Although much is known about the molecular mechanisms involved in transporting soluble proteins to the central vacuole, the mechanisms governing the trafficking of membrane proteins remain largely unknown. In this study, we investigated the mechanism involved in targeting the membrane protein, AtβFructosidase 4 (AtβFruct4), to the central vacuole in protoplasts. AtβFruct4 as a green fluorescent protein (GFP) fusion protein was transported as a membrane protein during transit from the endoplasmic reticulum (ER) through the Golgi apparatus and the prevacuolar compartment (PVC). The N-terminal cytosolic domain of AtβFruct4 was sufficient for transport from the ER to the central vacuole and contained sequence motifs required for trafficking. The sequence motifs, LL and PI, were found to be critical for ER exit, while the EEE and LCPYTRL sequence motifs played roles in trafficking primarily from the trans Golgi network (TGN) to the PVC and from the PVC to the central vacuole, respectively. In addition, actin filaments and AtRabF2a, a Rab GTPase, played critical roles in vacuolar trafficking at the TGN and PVC, respectively. On the basis of these results, we propose that the vacuolar trafficking of AtβFruct4 depends on multiple sequence motifs located at the N-terminal cytoplasmic domain that function as exit and/or sorting signals in different stages during the trafficking process.     

Rab26 Modulates the Cell Surface Transport of α2-Adrenergic Receptors from the Golgi

The molecular mechanisms underlying the transport from the Golgi to the cell surface of G protein-coupled receptors remain poorly elucidated. Here we determined the role of Rab26, a Ras-like small GTPase involved in vesicle-mediated secretion, in the cell surface export of α₂-adrenergic receptors. We found that transient expression of Rab26 mutants and siRNA-mediated depletion of Rab26 significantly attenuated the cell surface numbers of α_2A-AR and α_2B-AR, as well as ERK1/2 activation by α_2B-AR. Furthermore, the receptors were extensively arrested in the Golgi by Rab26 mutants and siRNA. Moreover, Rab26 directly and activation-dependently interacted with α_2B-AR, specifically the third intracellular loop. These data demonstrate that the small GTPase Rab26 regulates the Golgi to cell surface traffic of α₂-adrenergic receptors, likely through a physical interaction. These data also provide the first evidence implicating an important function of Rab26 in coordinating plasma membrane protein transport.     

Importin α Partitioning to the Plasma Membrane Regulates Intracellular Scaling

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Monocyte Trafficking,Engraftment, and Delivery of Nanoparticles and an Exogenous Gene into the Acutely Inflamed Brain Tissue – Evaluations on Monocyte-Based Delivery System for the Central Nervous System

The ability of monocytes and monocyte-derived macrophages (MDM) to travel towards chemotactic gradient, traverse tissue barriers, and accumulate precisely at diseased sites makes them attractive candidates as drug carriers and therapeutic gene delivery vehicles targeting the brain, where treatments are often hampered by the blockade of the blood brain barrier (BBB). This study was designed to fully establish an optimized cell-based delivery system using monocytes and MDM, by evaluating their homing efficiency, engraftment potential, as well as carriage and delivery ability to transport nano-scaled particles and exogenous genes into the brain, following the non-invasive intravenous (IV) cell adoptive transfer in an acute neuroinflammation mouse model induced by intracranial injection of Escherichia coli lipopolysaccharides. We demonstrated that freshly isolated monocytes had superior inflamed-brain homing ability over MDM cultured in the presence of macrophage colony stimulating factor. In addition, brain trafficking of IV infused monocytes was positively correlated with the number of adoptive transferred cells, and could be further enhanced by transient disruption of the BBB with IV administration of Mannitol, Bradykinin or Serotonin right before cell infusion. A small portion of transmigrated cells was detected to differentiate into IBA-1 positive cells with microglia morphology in the brain. Finally, with the use of superparamagnetic iron oxide nanoparticles SHP30, the ability of nanoscale agent-carriage monocytes to enter the inflamed brain region was validated. In addition, lentiviral vector DHIV-101 was used to introduce green fluorescent protein (GFP) gene into monocytes, and the exogenous GFP gene was detected in the brain at 48 hours following IV infusion of the transduced monocytes. All together, our study has set up the optimized conditions for the more-in-depth tests and development of monocyte-mediated delivery, and our data supported the notion to use monocytes as a non-invasive cell-based delivery system for the brain.     

Involvement of the Golgi apparatus in the secretion of α-amylase from gibberellin-treated barley aleurone cells

Localisation of -amylase (EC 3.2.1.1) in barley aleurone cells treated with gibberellic acid has been achieved using protein A-gold-labelled polyclonal antibodies. Gold particles were located almost exclusively over the lumen of the rough endoplasmic reticulum and cisternae of the Golgi apparatus. The label was most concentrated over the Golgi apparatus. This indicates that the Golgi is involved in the secretion of -amylase protein from aleurone cells.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - PBS phosphate-buffered saline     

Regulation of Constitutive Cargo Transport from the trans-Golgi Network to Plasma Membrane by Golgi-localized G Protein βγ Subunits

Observations of Golgi fragmentation upon introduction of G protein βγ (Gβγ) subunits into cells have implicated Gβγ in a pathway controlling the fission at the trans-Golgi network (TGN) of plasma membrane (PM)-destined transport carriers. However, the subcellular location where Gβγ acts to provoke Golgi fragmentation is not known. Additionally, a role for Gβγ in regulating TGN-to-PM transport has not been demonstrated. Here we report that constitutive or inducible targeting of Gβγ to the Golgi, but not other subcellular locations, causes phospholipase C- and protein kinase D-dependent vesiculation of the Golgi in HeLa cells; Golgi-targeted β₁γ₂ also activates protein kinase D. Moreover, the novel Gβγ inhibitor, gallein, and the Gβγ-sequestering protein, GRK2ct, reveal that Gβγ is required for the constitutive PM transport of two model cargo proteins, VSV-G and ss-HRP. Importantly, Golgi-targeted GRK2ct, but not a PM-targeted GRK2ct, also blocks protein transport to the PM. To further support a role for Golgi-localized Gβγ, endogenous Gβ was detected at the Golgi in HeLa cells. These results are the first to establish a role for Golgi-localized Gβγ in regulating protein transport from the TGN to the cell surface.     

Mechanism for Targeting the A-kinase Anchoring Protein AKAP18δ to the Membrane

A-kinase anchoring proteins (AKAPs) are a family of scaffolding proteins that target PKA and other signaling molecules to cellular compartments and thereby spatiotemporally define cellular signaling events. The AKAP18 family comprises AKAP18α, AKAP18β, AKAP18γ, and AKAP18δ. The δ isoform targets PKA and phosphodiesterase PDE4D to AQP2 (aquaporin-2)-bearing vesicles to orchestrate the acute regulation of body water balance. Therefore, AKAP18δ must adopt a membrane localization that seems at odds with (i) its lack of palmitoylation or myristoylation sites that tailor its isoforms AKAP18α and AKAP18β to membrane compartments and (ii) the high sequence identity to the preferentially cytoplasmic AKAP18γ. Here, we show that the electrostatic attraction of the positively charged amino acids of AKAP18δ to negatively charged lipids explains its membrane targeting. As revealed by fluorescence correlation spectroscopy, the binding constant of purified AKAP18δ fragments to large unilamellar vesicles correlates (i) with the fraction of net negatively charged lipids in the bilayer and (ii) with the total amount of basic residues in the protein. Although distantly located on the sequence, these positively charged residues concentrate in the tertiary structure and form a clear binding surface. Thus, specific recruitment of the AKAP18δ-based signaling module to membranes such as those of AQP2-bearing vesicles must be achieved by additional mechanisms, most likely compartment-specific protein-protein interactions.     

Using Ultrasonography to Define Fetal�CMaternal Relationships: Moving from Humans to Mice

Ultrasound scanning is a noninvasive, accurate, and cost-effective method to create images of the female reproductive tract clinically and in research. Ultrasonography is particularly valuable for studying the dynamic relationships among mother, placenta, and fetus during pregnancy because this modality does not disturb the ongoing course of gestation. Importantly, the complex vascular changes in the mother induced by pregnancy and the vascular system generated to support placental function can be assessed quantitatively and functionally by ultrasonography. Many mouse models are available that address aspects of human placental function and dysfunction, but high-quality microultrasound technology suitable for use in pregnant mice has become widely available only recently. This technical advance now enables real-time recording of maternal–fetal interactions in pregnant rodents. The ability to perform microultrasonic analyses of parameters such as uterine arterial remodeling, hemodynamic changes, placental development, and fetal growth in mice now permits research that uses the same imaging platform as that for human patients. This capability will enhance the translation of information derived from rodent studies to the clinic.Abbreviations: gd, gestation day; IUGR, intrauterine growth restrictionHuman pregnancy is a complex, dynamic progress accompanied by both inspiring and amazing events. Clinically, disorders of pregnancy are among the least understood areas in health science because of ethical and cultural challenges and the involvement of 2 (or more) subjects (mother and fetus) rather than 1. Because continuation and successful conclusion of pregnancy typically are desired, advances in clinical and research investigations of human pregnancy are highly dependent upon technological advances in noninvasive imaging. The most common human gestational complications are preeclampsia (acute onset of hypertension during the second half of pregnancy), intrauterine growth restriction (IUGR), and diabetes. These pathologies are thought to share many risk factors, particularly an inadequate response of the maternal vasculature to the state of pregnancy.⁵⁹ In all mammals, maternal vascular adaptation is essential to meet the requirements of blastocyst implantation and placental and fetal development to ensure a successful pregnancy. In species with hemochorial placentation (including human, rat, and mouse), endometrial decidualization is associated with maternal vascular remodeling at implantation sites, and the remodeling process has become a diagnostic and therapeutic target.^39,47Ultrasonography was introduced in 1958 and has become the diagnostic tool of choice to study human gestational health,¹⁸ not only because this modality is noninvasive with very low risk but also because it provides real-time images with positional information.²⁴ Ultrasonography is relatively inexpensive and portable, compared with other modalities such as MRI and computed tomography. Modern clinical ultrasonography usually is performed by using a pulse–echo approach with a brightness-mode (B-mode) output that can be augmented by using advanced functions such as color Doppler mapping, 3D or 4D imaging, and spectral measurements. Application of these ultrasound techniques has enabled exploration and understanding of human maternal–fetal relationships that previously had been virtually inaccessible. Most studies of the physiology and pathology of pregnancy in animal models still rely on postmortem approaches rather than serial examinations, which would decrease experimental animal numbers. A key advance for studies of pregnancy in mice has been the development of microultrasonography. This technology now provides in vivo morphometric quantification of embryonic and placental growth for mice.^21,33 Here we review the application of microultrasonography to hemodynamic monitoring, vascular assessment, and placental development in mice and address its limitations briefly.     

Identification of β-Lactamase Inhibitory Peptide Using Yeast Two-Hybrid System

Random oligonucleotide fragments were designed and amplified by PCR and fused with the activating domain of pGAD424 to construct a random peptide library. The DNA fragment encoding beta-lactamase was fused with the binding domain of pGBT9(+2). Subsequently, using yeast two-hybrid system we found two positive clones encoding peptides P1 and P2 that have the ability to bind beta-lactamase in vivo. The genes encoding P1 and P2 were cloned into pGEX-4T-1. GST-peptide fusion proteins were expressed in Escherichia coli and isolated by glutathione-Sepharose 4B affinity chromatography. Finally, P1 and P2 were cleaved from the fusion protein with thrombin and purified by ultrafiltration. Inhibition assay of peptides with beta-lactamase in vitro indicated that only P1 has the ability to inhibit beta-lactamase.