Entry at the trans-Face of the Golgi

The trans-Golgi network (TGN) receives a select set of proteins from the endocytic pathway—about 5% of total plasma membrane glycoproteins (Duncan and Kornfeld 1988). Proteins that are delivered include mannose 6-phosphate receptors (MPRs), TGN46, sortilin, and various toxins that hitchhike a ride backward through the secretory pathway to intoxicate cells after they exit into the cytoplasm from the endoplasmic reticulum (ER). This article will review work on the molecular players that drive protein transport from the endocytic pathway to the TGN. Distinct requirements have revealed multiple routes for retrograde transport; in addition, the existence of multiple, potential coat proteins and/or cargo adaptors imply that multiple vesicular transfers are likely involved. Several comprehensive reviews have appeared recently and should be sought for additional details (Bonifacino and Rojas 2006; Johannes and Popoff 2008).     

Sphingomyelin-enriched Microdomains at the Golgi Complex

Sphingomyelin- and cholesterol-enriched microdomains can be isolated as detergent-resistant membranes from total cell extracts (total-DRM). It is generally believed that this total-DRM represents microdomains of the plasma membrane. Here we describe the purification and detailed characterization of microdomains from Golgi membranes. These Golgi-derived detergent-insoluble complexes (GICs) have a low buoyant density and are highly enriched in lipids, containing 25% of total Golgi phospholipids including 67% of Golgi-derived sphingomyelin, and 43% of Golgi-derived cholesterol. In contrast to total-DRM, GICs contain only 10 major proteins, present in nearly stoichiometric amounts, including the alpha- and beta-subunits of heterotrimeric G proteins, flotillin-1, caveolin, and subunits of the vacuolar ATPase. Morphological data show a brefeldin A-sensitive and temperature-sensitive localization to the Golgi complex. Strikingly, the stability of GICs does not depend on its membrane environment, because, after addition of brefeldin A to cells, GICs can be isolated from a fused Golgi-endoplasmic reticulum organelle. This indicates that GIC microdomains are not in a dynamic equilibrium with neighboring membrane proteins and lipids. After disruption of the microdomains by cholesterol extraction with cyclodextrin, a subcomplex of several GIC proteins including the B-subunit of the vacuolar ATPase, flotillin-1, caveolin, and p17 could still be isolated by immunoprecipitation. This indicates that several of the identified GIC proteins localize to the same microdomains and that the microdomain scaffold is not required for protein interactions between these GIC proteins but instead might modulate their affinity.     

口岸出入境人员预防接种统计分析

目的:对口岸出入境人员的预防接种情况进行统计分析,为各种跨国传染性疾病的预防提供参考数据.方法:选择我处2010年1月至2012年5月口岸的出入境人员为研究对象,将其按公务人员、船员、劳务人员、留学人员、旅游探亲及商务等进行分组,并对其预防接种麻风腮(MMR)、ACYW群脑膜炎球菌多糖疫苗、黄热减毒活疫苗、水痘等9种疫苗的接种情况进行分析.结果:研究期间共为6870位出入境人员进行了预防接种,其中留学人员和劳务人员占有的比例较大;接种黄热减毒活疫苗和青蒿琥酯片的人数居多.结论:通过加强旅行疾病及预防接种相关知识的宣传,提高出入境人员预防接种率,可以降低各种传染性疾病通过口岸传染的几率,确保出入境人员的健康.     

An Assay for Clogging the Ciliary Pore Complex Distinguishes Mechanisms of Cytosolic and Membrane Protein Entry

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Mechanisms of protein retention in the Golgi

The protein composition of the Golgi is intimately linked to its structure and function. As the Golgi serves as the major protein-sorting hub for the secretory pathway, it faces the unique challenge of maintaining its protein composition in the face of constant influx and efflux of transient cargo proteins. Much of our understanding of how proteins are retained in the Golgi has come from studies on glycosylation enzymes, largely because of the compartment-specific distributions these proteins display. From these and other studies of Golgi membrane proteins, we now understand that a variety of retention mechanisms are employed, the majority of which involve the dynamic process of iterative rounds of retrograde and anterograde transport. Such mechanisms rely on protein conformation and amino acid-based sorting signals as well as on properties of transmembrane domains and their relationship with the unique lipid composition of the Golgi.     

Signaling Circuits on the Golgi Complex

Numerous components of signaling pathways involved in key cellular processes reside on the Golgi complex. Here, we will focus on the roles of signaling proteins that regulate cargo trafficking along the anterograde and retrograde pathways. Emphasis will also be put on the effects of these regulatory proteins on the maintenance of the structure and function of the Golgi, and in particular on the phosphorylation of key components of the transport machinery. These pathways position the Golgi complex as a central hub in the regulation of cell signaling. To date, however, the activation and coordination of these signaling molecules remain a mystery. Being able to describe the interplay between several of these signaling pathways and secretion, and the flow of information through these pathways, will help us to understand how the secretory machinery works and how it interacts with other cellular functions. This will also advance our understanding of how the secretory pathway functions under physiological circumstances, and how its dysregulation can initiate pathological conditions.     

Simultaneous Measurement of Smoothened Entry Into and Exit From the Primary Cilium

Ciliary accumulation of signaling proteins must result from a rate of ciliary entry that exceeds ciliary exit, but approaches for distinguishing ciliary entry vs. exit are lacking. Using a photoconvertible fluorescent protein tag, we establish an assay that allows a separate but simultaneous examination of ciliary entry and exit of the Hedgehog signaling protein Smoothened in individual cells. We show that KAAD-cyclopamine selectively blocks entry, whereas ciliobrevin interferes initially with exit and eventually with both entry and exit of ciliary Smoothened. Our study provides an approach to understanding regulation of ciliary entry vs. exit of Hedgehog signaling components as well as other ciliary proteins.     

Microtubule Cytoskeleton Remodeling by Acentriolar Microtubule-organizing Centers at the Entry and Exit from Mitosis in Drosophila Somatic Cells

Cytoskeleton microtubules undergo a reversible metamorphosis as cells enter and exit mitosis to build a transient mitotic spindle required for chromosome segregation. Centrosomes play a dominant but dispensable role in microtubule (MT) organization throughout the animal cell cycle, supporting the existence of concurrent mechanisms that remain unclear. Here we investigated MT organization at the entry and exit from mitosis, after perturbation of centriole function in Drosophila S2 cells. We found that several MTs originate from acentriolar microtubule-organizing centers (aMTOCs) that contain γ-tubulin and require Centrosomin (Cnn) for normal architecture and function. During spindle assembly, aMTOCs associated with peripheral MTs are recruited to acentriolar spindle poles by an Ncd/dynein-dependent clustering mechanism to form rudimentary aster-like structures. At anaphase onset, down-regulation of CDK1 triggers massive formation of cytoplasmic MTs de novo, many of which nucleated directly from aMTOCs. CDK1 down-regulation at anaphase coordinates the activity of Msps/XMAP215 and the kinesin-13 KLP10A to favor net MT growth and stability from aMTOCs. Finally, we show that microtubule nucleation from aMTOCs also occurs in cells containing centrosomes. Our data reveal a new form of cell cycle–regulated MTOCs that contribute for MT cytoskeleton remodeling during mitotic spindle assembly/disassembly in animal somatic cells, independently of centrioles.     

Signaling at the Golgi

The protein processing and trafficking function of the Golgi is intimately linked to multiple intracellular signaling pathways. Assembly of Golgi trafficking structures and lipid sorting at the Golgi complex is controlled and coordinated by specific phosphoinositide kinases and phosphatases. The intra-Golgi transport machinery is also regulated by kinases belonging to several functionally distinct families, for example, MAP kinase signaling is required for mitotic disassembly of the Golgi. However, the Golgi plays an additional, prominent role in compartmentalizing other signaling cascades that originate at the plasma membrane or at other organelles. This article summarizes recent advances in our understanding of the signaling network that converges at the Golgi.The Golgi apparatus is a dynamic structure that constantly exchanges proteins and lipids with other organelles. It is critical for organellar homeostasis that the different trafficking routes at the Golgi are precisely regulated. For example, the sorting and transport functions of the Golgi must be correctly coordinated with the overall activity of the secretory pathway. In addition, changes in Golgi structure and morphology are tightly controlled, which is particularly critical during mitosis, when the Golgi complex becomes disassembled for proper distribution between the dividing cells. It is therefore not surprising that diverse sets of signaling factors localize at the Golgi and control its function and shape.Phosphoinositide lipids have emerged as particularly important regulators of Golgi function. Reversible phosphorylation of the inositol headgroup of phosphatidylinositol creates seven distinct phosphoinositide species (Di Paolo and De Camilli 2006). These molecules serve as signal transducers at virtually every cellular membrane but have a particularly important role in controlling membrane traffic (Di Paolo and De Camilli 2006). A critical property of phosphoinositides is their tightly regulated spatial distribution. Recent studies have uncovered concentrated pools of these lipids at individual membranes including the Golgi (Roy and Levine 2004; De Matteis et al. 2005; Varnai and Balla 2008). Phosphoinositides often act in cooperation with small Ras-type GTPases and the interplay between phosphoinositides and GTPases from the ADP-ribosylation factor (Arf) and Ras-related in brain (Rab) families is essential for Golgi function (Behnia and Munro 2005; Mayinger 2009). How the lipid kinases and phosphatases that regulate Golgi phosphoinositides interact with other signaling pathway remains a challenging area of research.Whereas phosphoinositide signaling pathways are mainly controlled via extracellular signals that transmit metabolic status and growth conditions, Golgi function can also be regulated by signals that originate at other secretory organelles. Enhanced biosynthesis and processing of secretory proteins at the ER induces the activation of a signaling network that modulates intra-Golgi traffic and overall capacity of secretion (Sallese et al. 2009).Finally, there is mounting evidence that the Golgi serves as an important signaling platform for numerous signaling cascades that originate at the plasma membrane. The discovery that components of the Ras and the protein kinase A (PKA) pathways reside at the Golgi indicates that this organelle plays an important role in compartmentalizing signal transduction pathways (Quatela and Philips 2006; Sallese et al. 2009). This article will review our current understanding of signaling at the Golgi and also highlight the relevance of these processes for human disease.     

Re'COG'nition at the Golgi

The conserved oligomeric Golgi (COG) complex co-ordinates retrograde vesicle transport within the Golgi. These vesicles maintain the distribution of glycosylation enzymes between the Golgi's cisternae, and therefore COG is intimately involved in glycosylation homeostasis. Recent years have greatly enhanced our knowledge of COG's composition, protein interactions, cellular function and most recently also its structure. The emergence of COG-dependent human glycosylation disorders gives particular relevance to these advances. The structural data have firmly placed COG in the family of multi-subunit tethering complexes that it shares with the exocyst, Dsl1 and Golgi-associated retrograde protein (GARP) complexes. Here, we review our knowledge of COG's involvement in vesicle tethering at the Golgi. In particular, we consider what this knowledge may add to our molecular understanding of vesicle tethering and how it impacts on the fine tuning of Golgi function, most notably glycosylation.     

Structure and Function of the Golgi Complex in Rice Cells: Characterization of Golgi Membrane Glycoproteins

The structure and synthesis of the saccharide chains of Golgimembrane glycoproteins in suspension-cultured rice (Oryza sativaL.) cells were studied. Peanut lectin (PNA) and Ulex europaeuslectin-I (UEA-I) have high affinity for typical O-linked saccharidechains and both recognized the saccharide chains of rice Golgimembrane glycoproteins. These glycoproteins were also sensitiveto alkali and to O-glycanase. These results indicate that theGolgi membrane glycoproteins have O-linked saccharide chains.Brefeldin A, a specific inhibitor of Golgi-mediated secretion,induced morphological changes in Golgi complexes and preventedthe synthesis of the saccharide chains of the membrane glycoproteinsthat could be recognized by PNA and UEA-I. These glycoproteinswere typically localized in all compartments of the Golgi complex.Monensin can arrest the transport of secretory proteins frommedial to trans Golgi compartments but did not affect the formationand localization of the Golgi membrane glycoproteins. Tunicamycin,an inhibitor of the synthesis of N-linked saccharide chains,did not inhibit the synthesis of the saccharide chains of theseGolgi membrane glycoproteins. These results strongly suggestthat the synthesis of O-linked saccharide chains of Golgi membraneglycoproteins is initiated in the cis Golgi compartment. (Received September 24, 1992; Accepted June 4, 1993)     

Requirements for ER-Arrest and Sequential Exit to the Golgi of Tomato Spotted Wilt Virus Glycoproteins

The envelope glycoproteins Gn and Gc are major determinants in the assembly of Tomato spotted wilt virus (TSWV) particles at the Golgi complex. In this article, the ER-arrest of singly expressed Gc and the transport of both glycoproteins to the Golgi upon co-expression have been analyzed. While preliminary results suggest that the arrest of Gc at the ER (endoplasmic reticulum) did not appear to result from improper folding, transient expression of chimeric Gc, in which the transmembrane domain (TMD) and/or cytoplasmic tail (CT) were swapped for those from Gn, showed that the TMD of Gn was sufficient to allow ER-exit and transport to the Golgi. Expression of both glycoproteins in the presence of overexpressed Sar1p-specific guanosine nucleotide exchange factor Sec 12p, resulted in ER-retention demonstrating that the viral glycoproteins are transported to the Golgi in a COPII (coat protein II)-dependent manner. Inhibition of ER-Golgi transport by brefeldin A (BFA) had a similar effect on the localization of Gn. However, inhibition of ER (endoplasmic reticulum) to Golgi transport of co-expressed Gc and Gn by overexpression of Sec 12p or by BFA revealed distinct localization patterns, i.e. diffuse ER localization versus concentration at specific spots.     

Actin acting at the Golgi

The organization, assembly and remodeling of the actin cytoskeleton provide force and tracks for a variety of (endo)membrane-associated events such as membrane trafficking. This review illustrates in different cellular models how actin and many of its numerous binding and regulatory proteins (actin and co-workers) participate in the structural organization of the Golgi apparatus and in trafficking-associated processes such as sorting, biogenesis and motion of Golgi-derived transport carriers.     

Nicotinamide Impairs Entry into and Exit from Meiosis I in Mouse Oocytes

Following exit from meiosis I, mammalian oocytes immediately enter meiosis II without an intervening interphase, accompanied by rapid reassembly of a bipolar spindle that maintains condensed chromosomes in a metaphase configuration (metaphase II arrest). Here we study the effect of nicotinamide (NAM), a non-competitive pan-sirtuin inhibitor, during meiotic maturation in mouse oocytes. Sirtuins are a family of seven NAD⁺-dependent deacetylases (Sirt1-7), which are involved in multiple cellular processes and are emerging as important regulators in oocytes and embryos. We found that NAM significantly delayed entry into meiosis I associated with delayed accumulation of the Cdk1 co-activator, cyclin B1. GVBD was also inhibited by the Sirt2-specific inhibitor, AGK2, and in a very similar pattern to NAM, supporting the notion that as in somatic cells, NAM inhibits sirtuins in oocytes. NAM did not affect subsequent spindle assembly, chromosome alignment or the timing of first polar body extrusion (PBE). Unexpectedly, however, in the majority of oocytes with a polar body, chromatin was decondensed and a nuclear structure was present. An identical phenotype was observed when flavopiridol was used to induce Cdk1 inactivation during late meiosis I prior to PBE, but not if Cdk1 was inactivated after PBE when metaphase II arrest was already established, altogether indicating that NAM impaired establishment rather than maintenance of metaphase II arrest. During meiosis I exit in NAM-treated medium, we found that cyclin B1 levels were lower and inhibitory Cdk1 phosphorylation was increased compared with controls. Although activation of the anaphase-promoting complex-Cdc20 (APC-Cdc20) occurred on-time in NAM-treated oocytes, Cdc20 levels were higher in very late meiosis I, pointing to exaggerated APC-Cdc20-mediated proteolysis as a reason for lower cyclin B1 levels. Collectively, therefore, our data indicate that by disrupting Cdk1 regulation, NAM impairs entry into meiosis I and the establishment of metaphase II arrest.     

Role of Microtubules in the Organization of the Golgi Complex

The Golgi complex of mammalian cells is composed of cisternal stacks that function in processing and sorting of membrane and luminal proteins during transport from the site of synthesis in the endoplasmic reticulum to lysosomes, secretory vacuoles, and the cell surface. Even though exceptions are found, the Golgi stacks are usually arranged as an interconnected network in the region around the centrosome, the major organizing center for cytoplasmic microtubules. A close relation thus exists between Golgi elements and microtubules (especially the stable subpopulation enriched in detyrosinated and acetylated tubulin). After drug-induced disruption of microtubules, the Golgi stacks are disconnected from each other, partly broken up, dispersed in the cytoplasm, and redistributed to endoplasmic reticulum exit sites. Despite this, intracellular protein traffic is only moderately disturbed. Following removal of the drugs, scattered Golgi elements move along reassembling microtubules back to the centrosomal region and reunite into a continuous system. The microtubule-dependent motor proteins cytoplasmic dynein and kinesin bind to Golgi membranes and have been implicated in vesicular transport to and from the Golgi complex. Microinjection of dynein heavy chain antibodies causes dispersal of the Golgi complex, and the Golgi complex of cells lacking cytoplasmic dynein is likewise spread throughout the cytoplasm. In a similar manner, kinesin antibodies have been found to inhibit Golgi-to-endoplasmic reticulum transport in brefeldin A-treated cells and scattering of Golgi elements along remaining microtubules in cells exposed to a low concentration of nocodazole. The molecular mechanisms in the interaction between microtubules and membranes are, however, incompletely understood. During mitosis, the Golgi complex is extensively reorganized in order to ensure an equal partitioning of this single-copy organelle between the daughter cells. Mitosis-promoting factor, a complex of cdc2 kinase and cyclin B, is a key regulator of this and other events in the induction of cell division. Cytoplasmic microtubules depolymerize in prophase and as a result thereof, the Golgi stacks become smaller, disengage from each other, and take up a perinuclear distribution. The mitotic spindle is thereafter put together, aligns the chromosomes in the metaphase plate, and eventually pulls the sister chromatids apart in anaphase. In parallel, the Golgi stacks are broken down into clusters of vesicles and tubules and movement of protein along the exocytic and endocytic pathways is inhibited. Using a cell-free system, it has been established that the fragmentation of the Golgi stacks is due to a continued budding of transport vesicles and a concomitant inhibition of the fusion of the vesicles with their target membranes. In telophase and after cytokinesis, a Golgi complex made up of interconnected cisternal stacks is recreated in each daughter cell and intracellular protein traffic is resumed. This restoration of a normal interphase morphology and function is dependent on reassembly of a radiating array of cytoplasmic microtubules along which vesicles can be carried and on reactivation of the machinery for membrane fusion.     

A Single Point Mutation Controls the Cholesterol Dependence of Semliki Forest Virus Entry and Exit

Membrane fusion and budding are key steps in the life cycle of all enveloped viruses. Semliki Forest virus (SFV) is an enveloped alphavirus that requires cellular membrane cholesterol for both membrane fusion and efficient exit of progeny virus from infected cells. We selected an SFV mutant, srf-3, that was strikingly independent of cholesterol for growth. This phenotype was conferred by a single amino acid change in the E1 spike protein subunit, proline 226 to serine, that increased the cholesterol independence of both srf-3 fusion and exit. The srf-3 mutant emphasizes the relationship between the role of cholesterol in membrane fusion and virus exit, and most significantly, identifies a novel spike protein region involved in the virus cholesterol requirement.     

COG6 Interacts with a Subset of the Golgi SNAREs and Is Important for the Golgi Complex Integrity

Vesicular tethers and SNAREs are two key protein components that govern docking and fusion of intracellular membrane carriers in eukaryotic cells. The conserved oligomeric Golgi (COG) complex has been specifically implicated in the tethering of retrograde intra‐Golgi vesicles. Using yeast two‐hybrid and co‐immunoprecipitation approaches, we show that the COG6 subunit of the COG complex is capable of interacting with a subset of Golgi SNAREs, namely STX5, STX6, GS27 and SNAP29. Interaction with SNAREs is accomplished via the universal SNARE‐binding motif of COG6. Overexpression of COG6, or its depletion from cells, disrupts the integrity of the Golgi complex. Importantly, COG6 protein lacking the SNARE‐binding domain is deficient in Golgi binding, and is not capable of inducing Golgi complex fragmentation when overexpressed. These results indicate that COG6–SNARE interactions are important for both COG6 localization and Golgi integrity .