Structural-functional organization of Golgi apparatus

This review is dedicated to the structure and function of Golgi apparatus (GA). It summarizes contemporary data published in numerous experimental papers and in several reviews. Possible ways of intra-Golgi transport of proteins, existent models of structural and functional organization of Golgi organelle, as well as the issues of its biogenesis, posttranslational modification and sorting of proteins and lipids, and mechanisms of their traffic-king are discussed. Special attention is paid to the role of coatomer proteins (COPI, COPII and clathrin), fusion proteins (SNAREs), and small GTPases (ARF, SARI) in the secretory pathway. In addition, the phenomena of ultrastructural alterations of GA due to various functional conditions and physiological stimuli are specifically accented. We included in this review our original data on a probable involvement of GA in water transport, and on the organization of atypical GA in microsporidia--intracellular parasitic protists.     

New components of the Golgi matrix

The eukaryotic Golgi apparatus is characterized by a stack of flattened cisternae that are surrounded by transport vesicles. The organization and function of the Golgi require Golgi matrix proteins, including GRASPs and golgins, which exist primarily as fiber-like bridges between Golgi cisternae or between cisternae and vesicles. In this review, we highlight recent findings on Golgi matrix proteins, including their roles in maintaining the Golgi structure, vesicle tethering, and novel, unexpected functions. These new discoveries further our understanding of the molecular mechanisms that maintain the structure and the function of the Golgi, as well as its relationship with other cellular organelles such as the centrosome.     

Intra-Golgi protein transport depends on a cholesterol balance in the lipid membrane

Transport of proteins between intracellular membrane compartments is mediated by a protein machinery that regulates the budding and fusion processes of individual transport steps. Although the core proteins of both processes are defined at great detail, much less is known about the involvement of lipids. Here we report that changing the cellular balance of cholesterol resulted in changes of the morphology of the Golgi apparatus, accompanied by an inhibition of protein transport. By using a well characterized cell-free intra-Golgi transport assay, these observations were further investigated, and it was found that the transport reaction is sensitive to small changes in the cholesterol content of Golgi membranes. Addition as well as removal of cholesterol (10 +/- 6%) to Golgi membranes by use of methyl-beta-cyclodextrin specifically inhibited the intra-Golgi transport assay. Transport inhibition occurred at the fusion step. Modulation of the cholesterol content changed the lipid raft partitioning of phosphatidylcholine and heterotrimeric G proteins, but not of other (non) lipid raft proteins and lipids. We suggest that the cholesterol balance in Golgi membranes plays an essential role in intra-Golgi protein transport and needs to be carefully regulated to maintain the structural and functional organization of the Golgi apparatus.     

Membrane trafficking in higher plant cells: GFP and antibodies, partners for probing the secretory pathway.

Eukaryotic cells are characterised by the organised distribution of membrane bounded compartments in their cytoplasm. The endoplasmic reticulum (ER) and the Golgi apparatus (GA) are part of this endomembrane machinery. They are involved in protein flow, and are in charge of specific functions such as the assembly, sorting and transport of newly synthesised proteins, glycoproteins or polysaccharides to their final destination, where the macromolecules are recognised either for action, storage, deposition or degradation. The structural and functional relationship between the ER and GA in higher plants is still a matter of debate. Therefore, it was essential to develop probes that would specifically label proteins or glycoproteins of the endomembrane system in situ. Here we compare two complementary approaches to probe plant endomembranes; immunocytochemistry on fixed cells, and in vivo studies using the expression of GFP tagged chimeric proteins. The structural relationship between ER and GA as based on pharmacological approaches using the two systems is explored.     

Characterization of Golgi scaffold proteins and their roles in compartmentalizing cell signaling

Subcellular compartmentalization has become an important theme in cell signaling. In particular, the Golgi apparatus (GA) plays a prominent role in compartmentalizing signaling cascades that originate at the plasma membrane or other organelles. To precisely regulate this process, cells have evolved a unique class of organizer proteins, termed “scaffold proteins”. Sef, PAQR3, PAQR10 and PAQR11 are scaffold proteins that have recently been identified on the GA and are referred to as Golgi scaffolds. The major cell growth signaling pathways, such as Ras/MAPK, PI3K/AKT, insulin and VEGF (vascular endothelial growth factor), are tightly regulated spatially and temporally by these Golgi scaffolds to ensure a physiologically appropriate outcome. Here, we discuss the subcellular localization and characterization of the topology and functional domains of these Golgi scaffolds and summarize their roles in the compartmentalization of cell signaling. We also highlight the physiological and pathological roles of these Golgi scaffolds in tumorigenesis and developmental disorders.     

Protein transport in plant cells: in and out of the Golgi

In plant cells, the Golgi apparatus is the key organelle for polysaccharide and glycolipid synthesis, protein glycosylation and protein sorting towards various cellular compartments. Protein import from the endoplasmic reticulum (ER) is a highly dynamic process, and new data suggest that transport, at least of soluble proteins, occurs via bulk flow. In this Botanical Briefing, we review the latest data on ER/Golgi inter-relations and the models for transport between the two organelles. Whether vesicles are involved in this transport event or if direct ER-Golgi connections exist are questions that are open to discussion. Whereas the majority of proteins pass through the Golgi on their way to other cell destinations, either by vesicular shuttles or through maturation of cisternae from the cis- to the trans-face, a number of membrane proteins reside in the different Golgi cisternae. Experimental evidence suggests that the length of the transmembrane domain is of crucial importance for the retention of proteins within the Golgi. In non-dividing cells, protein transport out of the Golgi is either directed towards the plasma membrane/cell wall (secretion) or to the vacuolar system. The latter comprises the lytic vacuole and protein storage vacuoles. In general, transport to either of these from the Golgi depends on different sorting signals and receptors and is mediated by clathrin-coated and dense vesicles, respectively. Being at the heart of the secretory pathway, the Golgi (transiently) accommodates regulatory proteins of secretion (e.g. SNAREs and small GTPases), of which many have been cloned in plants over the last decade. In this context, we present a list of regulatory proteins, along with structural and processing proteins, that have been located to the Golgi and the 'trans-Golgi network' by microscopy.     

The plant ER-Golgi interface: a highly structured and dynamic membrane complex

As compared with other eukaryotic cells, plants have developed an endoplasmic reticulum (ER)-Golgi interface with very specific structural characteristics. ER to Golgi and Golgi to ER transport appear not to be dependent on the cytoskeleton, and ER export sites have been found closely associated with Golgi bodies to constitute entire mobile units. However, the molecular machinery involved in membrane trafficking seems to be relatively conserved among eukaryotes. Therefore, a challenge for plant scientists is to determine how these molecular machineries work in a different structural and dynamic organization. This review will focus on some aspects of membrane dynamics that involve coat proteins, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment receptor proteins), lipids, and lipid-interacting proteins.     

Role of the Golgi Apparatus in the Blood-Brain Barrier: Golgi Protection May Be a Targeted Therapy for Neurological Diseases

The blood-brain barrier (BBB) protects the brain from toxic material in the blood, provides nutrients for brain tissues, and screens harmful substances from the brain. The specific brain microvascular endothelial cells (BMVECs), tight junction between endothelial cells, and astrocytes ensure proper function of the central nervous system (CNS). Pathological factors disrupt the integrity of the BBB by destroying the normal function of endothelial cells and decreasing the production of tight junction proteins or the expression of proteins specifically localized on astrocytes. Interestingly, fragmentation of the Golgi apparatus is observed in neurological diseases and is involved in the destruction of the BBB function. The Golgi acts as a processing center in which proteins are transported after being processed in the endoplasmic reticulum. Besides reprocessing, classifying, and packaging proteins, the Golgi apparatus (GA) also acts as a signaling platform and calcium pool. In this review, we summarized the current literature on the potential relationship between the Golgi and endothelial cells, tight junction, and astrocytes. The normal function of the BBB is maintained as long as the normal function and morphology of the GA are not disturbed. Furthermore, we speculate that protecting the Golgi may be a novel therapeutic approach to protect the BBB and treat neurological diseases due to BBB dysfunction.     

Sphingolipid transport in mitotic HeLa cells.

Mitotic and interphase HeLa cells were labeled with [3H]serine. Ceramide and its derivatives, lactosylceramide and sphingomyelin, were biosynthetically labeled under both conditions. Only in the absence of nocodazole, as the cells entered telophase, was an additional glycosphingolipid synthesized which was identified as GA2 (GalNAc(beta 1,4)Gal(beta 1,4)Glc(beta 1,1)Cer). Ceramide, the basic sphingolipid precursor, is synthesized in the endoplasmic reticulum, whereas its immediate derivatives are synthesized in early Golgi compartments. Transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi is inhibited in mitotic cells while ceramide acquires early Golgi modifications under the same conditions, suggesting that ceramide can be delivered to the Golgi by a different route. Since GA2 is synthesized in late Golgi, its absence in mitotic cells strongly argues for an in vivo inhibition of intra-Golgi transport, an observation with important implications for the mechanism of Golgi division.     

Organization of SNAREs within the Golgi Stack

Antero- and retrograde cargo transport through the Golgi requires a series of membrane fusion events. Fusion occurs at the cis- and trans-side and along the rims of the Golgi stack. Four functional SNARE complexes have been identified mediating lipid bilayer merger in the Golgi. Their function is tightly controlled by a series of reactions involving vesicle tethering and SM proteins. This network of protein interactions spatially and temporally determines the specificity of transport vesicle targeting and fusion within the Golgi.At steady state, the Golgi maintains its structural and functional organization despite a massive lipid and protein flow. A balanced anterograde and retrograde membrane flow are required to constantly recycle the transport machinery and cargo containers (vesicles). In the absence of efficient recycling, directional net cargo transport would cease and the Golgi would collapse. Thus, transport vesicles constantly leave and enter at both sides of the Golgi stack and bud and fuse along the rims of the cisternae. To maintain the compartmental identity, vesicle fusion occurs in a specific and orchestrated manner. These fusion events are mediated by a cascade of reactions centered around the membrane fusion proteins SNAREs (SNAP receptors) (Söllner et al. 1993b).     

Src regulates Golgi structure and KDEL receptor-dependent retrograde transport to the endoplasmic reticulum

The tyrosine kinase Src is present on the Golgi membranes. Its role, however, in the overall function and organization of the Golgi apparatus is unclear. We have found that in a cell line called SYF, which lacks the three ubiquitous Src-like kinases (Src, Yes, and Fyn), the organization of the Golgi apparatus is perturbed. The Golgi apparatus is composed of collapsed stacks and bloated cisternae in these cells. Expression of an activated form of Src relocated the KDEL receptor (KDEL-R) from the Golgi apparatus to the endoplasmic reticulum. Other Golgi-specific marker proteins were not affected under these conditions. Because of the specific effect of Src on the location of KDEL-R, we tested whether protein transport between ER and the Golgi apparatus involves Src. Transport of Pseudomonas exotoxin, which is transported to the ER by binding to the KDEL-R is accelerated by inhibition or genetic ablation of Src. Protein transport from ER to the Golgi apparatus however, is unaffected by Src deletion or inhibition. We propose that Src has an appreciable role in the organization of the Golgi apparatus, which may be linked to its involvement in protein transport from the Golgi apparatus to the endoplasmic reticulum.     

Geldanamycin Enhances Retrograde Transport of Shiga Toxin in HEp-2 Cells

The heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA) has been shown to alter endosomal sorting, diverting cargo destined for the recycling pathway into the lysosomal pathway. Here we investigated whether GA also affects the sorting of cargo into the retrograde pathway from endosomes to the Golgi apparatus. As a model cargo we used the bacterial toxin Shiga toxin, which exploits the retrograde pathway as an entry route to the cytosol. Indeed, GA treatment of HEp-2 cells strongly increased the Shiga toxin transport to the Golgi apparatus. The enhanced Golgi transport was not due to increased endocytic uptake of the toxin or perturbed recycling, suggesting that GA selectively enhances endosomal sorting into the retrograde pathway. Moreover, GA activated p38 and both inhibitors of p38 or its substrate MK2 partially counteracted the GA-induced increase in Shiga toxin transport. Thus, our data suggest that GA-induced p38 and MK2 activation participate in the increased Shiga toxin transport to the Golgi apparatus.     

Coordinated lipid transfer between the endoplasmic reticulum and the Golgi complex requires the VAP proteins and is essential for Golgi-mediated transport

Lipid transport between intracellular organelles is mediated by vesicular and nonvesicular transport mechanisms and is critical for maintaining the identities of different cellular membranes. Nonvesicular lipid transport between the endoplasmic reticulum (ER) and the Golgi complex has been proposed to affect the lipid composition of the Golgi membranes. Here, we show that the integral ER-membrane proteins VAP-A and VAP-B affect the structural and functional integrity of the Golgi complex. Depletion of VAPs by RNA interference reduces the levels of phosphatidylinositol-4-phosphate (PI4P), diacylglycerol, and sphingomyelin in the Golgi membranes, and it leads to substantial inhibition of Golgi-mediated transport events. These effects are coordinately mediated by the lipid-transfer/binding proteins Nir2, oxysterol-binding protein (OSBP), and ceramide-transfer protein (CERT), which interact with VAPs via their FFAT motif. The effect of VAPs on PI4P levels is mediated by the phosphatidylinositol/phosphatidylcholine transfer protein Nir2, which is required for Golgi targeting of OSBP and CERT and the subsequent production of diacylglycerol and sphingomyelin. We propose that Nir2, OSBP, and CERT function coordinately at the ER-Golgi membrane contact sites, thereby affecting the lipid composition of the Golgi membranes and consequently their structural and functional identities.     

The Role of the Golgi-Resident SPCA Ca<Superscript>2+</Superscript>/Mn<Superscript>2+</Superscript> Pump in Ionic Homeostasis and Neural Function

Recent evidence highlights the functional importance of the Golgi apparatus (GA) in neurological diseases. The functions of the mammalian GA, in addition to the processing and transport of cargo, also include ionic homeostasis. Besides Ca²⁺-release channels which serves GA as an agonist-sensitive intracellular Ca²⁺ store, and Ca²⁺-binding proteins, the GA contains Ca²⁺-uptake mechanisms consisting of the well-known sarco-endoplasmic reticulum Ca²⁺-transport ATPases and the much less characterized secretory-pathway Ca²⁺-transport ATPases (SPCA). SPCA can transport both Ca²⁺ and Mn²⁺ into the Golgi lumen and therefore is involved in the cytosolic and intra-Golgi Ca²⁺ and Mn²⁺ homeostasis. It has shown that both of the mRNA and protein of SPCAs are highly expressed in brain. In addition, brain is the region with the highest activity of SPCA isoforms, which may be related to the involvement of Ca²⁺ and Mn²⁺ homeostasis in neural functions. In this review, we compile some recent findings showing that the SPCA isoform plays a much more important role in intracellular ionic homeostasis than previously anticipated and illustrating the involvement of SPCA isoforms in certain neurophysiological or neuropathological process. We are interested in gaining insight into the intricate role of the SPCA pumps to explain the GA-specific functions in neurological disorders.     

PKA-Mediated Golgi Remodeling During cAMP Signal Transmission

Cyclic AMP (cAMP)-dependent protein kinase A (PKA) is part of the set of signaling proteins that are stably associated to the cytosolic surface of Golgi membranes in mammalian cells. In principle, Golgi-associated PKA could participate in either signal transduction events and/or the coordination of Golgi transport activities. Here, we show data indicating that although Golgi-associated PKA is activated fast and efficiently during cell stimulation by an extracellular ligand it does not contribute significantly to cAMP signal transmission to the nucleus. Instead, most of the PKA catalytic subunits C α derived from the Golgi complex remain localized in the perinuclear cytoplasm where they induce changes in Golgi structural organization. Thus, in stimulated cells the Golgi complex appears collapsed, showing increased colocalization of previously segregated markers and exhibiting merging of different proximal cisternae within a single stack. In contrast, the trans -Golgi network remains as a separate compartment. Consequently, the rate of protein transport is increased whereas glycan processing is not severely affected. This remodeling process requires the presence of PKA activity associated to the Golgi membranes. Together these data indicate that Golgi-associated PKA activity is involved in the adaptation of Golgi dynamic organization to extracellular signaling events.     

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.     

The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells

The organization of intracellular compartments and the transfer of components between them are central to the correct functioning of mammalian cells. Proteins and lipids are transferred between compartments by the formation, movement and subsequent specific fusion of transport intermediates. These vesicles and membrane clusters must be coupled to the cytoskeleton and to motor proteins that drive motility. Anterograde ER (endoplasmic reticulum)-to-Golgi transport, and the converse step of retrograde traffic from the Golgi to the ER, are now known to involve coupling of membranes to the microtubule cytoskeleton. Here we shall discuss our current understanding of the mechanisms that link membrane traffic in the early secretory pathway to the microtubule cytoskeleton in mammalian cells. Recent data have also provided molecular detail of functional co-ordination of motor proteins to specify directionality, as well as mechanisms for regulating motor activity by protein phosphorylation.     

Developmental regulation of membrane traffic organization during synaptogenesis in mouse diaphragm muscle

In innervated adult skeletal muscles, the Golgi apparatus (GA) displays a set of remarkable features in comparison with embryonic myotubes. We have previously shown by immunocytochemical techniques, that in adult innervated fibers, the GA is no longer associated with all the nuclei, but appears to be concentrated mostly in the subneural domain under the nerve endings in chick (Jasmin, B. J., J. Cartaud, M. Bornens, and J.- P. Changeux. 1989. Proc. Natl. Acad. Sci. USA. 86:7218-7222) and rat (Jasmin, B. J., C. Antony, J.-P. Changeux, and J. Cartaud. 1995. Eur. J. Neurosci. 7:470-479). In addition to such compartmentalization, biochemical modifications take place that suggest a functional specialization of the subsynaptic GA. Here, we focused on the developmental regulation of the membrane traffic organization during the early steps of synaptogenesis in mouse diaphragm muscle. We investigated by immunofluorescence microscopy on cryosections, the distribution of selected subcompartments of the exocytic pathway, and also of a representative endocytic subcompartment with respect to the junctional or extrajunctional domains of developing myofibers. We show that throughout development the RER, the intermediate compartment, and the prelysosomal compartment (mannose 6-phosphate receptor-rich compartment) are homogeneously distributed along the fibers, irrespective of the subneural or extrajunctional domains. In contrast, at embryonic day E17, thus 2-3 d after the onset of innervation, most GA markers become restricted to the subneural domain. Interestingly, some Golgi markers (e.g., alpha-mannosidase II, TGN 38, present in the embryonic myotubes) are no longer detected in the innervated fiber even in the subsynaptic GA. These data show that in innervated muscle fibers, the distal part of the biosynthetic pathway, i.e., the GA, is remodeled selectively shortly after the onset of innervation. As a consequence, in the innervated fiber, the GA exists both as an evenly distributed organelle with basic functions, and as a highly differentiated subsynaptic organelle ensuring maturation and targeting of synaptic proteins. Finally, in the adult, denervation of a hemidiaphragm causes a burst of reexpression of all Golgi markers in extrasynaptic domains of the fibers, hence showing that the particular organization of the secretory pathway is placed under nerve control.     

Golgi apparatus fragmentation as a mechanism responsible for uniform delivery of uroplakins to the apical plasma membrane of uroepithelial cells

Background information. The GA (Golgi apparatus) has an essential role in membrane trafficking, determining the assembly and delivery of UPs (uroplakins) to the APM (apical plasma membrane) of superficial UCs (uroepithelial cells) of urinary bladder. UPs are synchronously and uniformly delivered from the GA to the APM by DFVs (discoidal‐ or fusiform‐shaped vesicles); however, the mechanism of UP delivery is not known. We have used the culture model of UCs with the capacity to undergo terminal differentiation to study the process of uniform delivery of DFVs to the APM and to elucidate the mechanisms involved. Results. By three‐dimensional localization using confocal microscopy of immunofluorescence‐labelled GA‐related markers [GM130 (cis‐Golgi matrix protein of 130 kDa), GS15 (Golgi Snare 15 kDa), GS28 and giantin], uroepithelial differentiation‐related markers (UPs), MTs (microtubules; α‐tubulin) and intermediate filaments [CK7 (cytokeratin 7) and CK20], we found that in non‐differentiated, UP‐negative UCs the GA is mostly organized as a single ribbon‐like structure close to the nucleus, whereas in differentiated, UP‐positive UCs the GA is fragmented and spread almost through the entire cell. The FRAP (fluorescence recovery after photobleaching) experiments on the UCs transfected with GalT (trans‐Golgi/TGN enzyme β1,4‐galactosyltransferase) fused to fluorescent protein showed that Golgi‐resident enzyme cycles freely within ribbon‐like GA but not within fragmented GA. By CLEM (correlative light—electron microscopy), we examined the GA fragments in cells expressing UPs. We found that GA fragments are fully functional and similar to the GA fragments that are formed after nocodazole treatment. Furthermore, we demonstrated that the reorganization of GA into a fragmented form is associated with the impairment of the MT organization in the basal, central and subapical cytoplasm and the accumulation of intermediate filaments in the apical cytoplasm that could affect the kinetics of MT star leading to the peripheral fragmentation of the GA in the differentiated UCs. Conclusions. The fragmentation of the GA and the subsequent spreading of GA to the cell periphery represent one of the key events that promote the uniform delivery of UPs over the entire APM of differentiating UCs and thus are of major importance in the final proper formation and maintenance of the blood—urine barrier.     

Spatial dissection of the cis- and trans-Golgi compartments in the malaria parasite Plasmodium falciparum

The Golgi apparatus forms the heart of the secretory pathway in eukaryotic cells where proteins are modified, processed and sorted. The transport of proteins from the endoplasmic reticulum (ER) to the cis- side of the Golgi complex takes place at specialized ER sub-domains known as transitional ER (tER). We used the Plasmodium falciparum orthologue of Sec13p to analyse tER organization. We show that the distribution of Pf Sec13p is restricted to defined areas of the ER membrane. These foci are juxtaposed to the Golgi apparatus and might represent tER sites. To further analyse cis - to trans -Golgi architecture, we generated a double transfectant parasite line that expresses the Golgi marker Golgi reassembly stacking protein (GRASP) as a green fluorescent protein fusion and the trans- Golgi marker Rab6 as a DsRed fusion protein. Our data demonstrate that Golgi multiplication is closely linked to tER multiplication, and that parasite maturation is accompanied by the spatial separation of the cis- and trans- face of this organelle.