Brefeldin A action and recovery in Chlamydomonas are rapid and involve fusion and fission of Golgi cisternae

CHLAMYDOMONAS NOCTIGAMA has a non-motile Golgi apparatus consisting of several Golgi stacks adjacent to transitional ER. These domains are characterized by vesicle-budding profiles and the lack of ribosomes on the side of the ER proximal to the Golgi stacks. Immunogold labelling confirms the presence of COPI-proteins at the periphery of the Golgi stacks, and COPII-proteins at the ER-Golgi interface. After addition of BFA (10 microg/ml) a marked increase in the number of vesicular profiles lying between the ER and the Golgi stacks is seen. Serial sections of cells do not provide any evidence for the existence of tubular connections between the ER and the Golgi stacks, supporting the notion that COPI- but not COPII-vesicle production is affected by BFA. The fusion of COPII-vesicles at the CIS-Golgi apparatus apparently requires the presence of retrograde COPI-vesicles. After 15 min the cisternae of neighbouring Golgi stacks begin to fuse forming "mega-Golgis", which gradually curl before fragmenting into clusters of vesicles and tubules. These are surrounded by the transitional ER on which vesicle-budding profiles are still occasionally visible. Golgi remnants continue to survive for several hours and do not completely disappear. Washing out BFA leads to a very rapid reassembly of Golgi cisternae. At first, clusters of vesicles are seen adjacent to transitional ER, then "mini Golgis" are seen whose cisternae grow in length and number to produce "mega Golgis". These structures then divide by vertical fission to produce Golgi stacks of normal size and morphology roughly 60 min after drug wash-out.     

Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway

Characteristics of brefeldin A (BFA)-induced redistribution of Golgi proteins into the endoplasmic reticulum (ER) and its relationship to an ER retrieval pathway were investigated. Retrograde movement of Golgi proteins into the ER occurred via long, tubulovesicular processes extending out of the Golgi along microtubules. Microtubule-disrupting agents (i.e., nocodazole), energy poisons, and reduced temperatures inhibited this pathway. In BFA-treated cells Golgi proteins appeared to cycle between the ER and an intermediate compartment marked by a 53 kd protein. Addition of nocodazole disrupted this dynamic cycle by preferentially inhibiting retrograde movement, causing Golgi proteins to accumulate in the intermediate compartment. In the absence of BFA, such an ER cycling pathway appeared to be followed normally by the 53 kd protein but not by Golgi proteins, as revealed by temperature shift experiments. We propose that BFA induces the interaction of the Golgi with an intermediate "recycling" compartment that utilizes a microtubule-dependent pathway into the ER.     

Golgi apparatus buds — vesicles or coated ends of tubules?

Summary Based on cell-free processing whereby membrane glycoproteins from one cell type were processed by enzymes located in Golgi apparatus from another cell type, J. Rothman and colleagues postulated that vesicles budding from one Golgi apparatus stack migrated to and fused with cisternal membranes of other Golgi apparatus stacks in the cell-free milieu. An extension of this hypothesis was that these same or similar vesicles were involved in the trafficking of membrane material from one cisterna to the next even in the same Golgi apparatus stack [W. G. Dunphy, J. E. Rothman: Compartmental organization of the Golgi stack. Cell 42: 13–21 (1985)]. A coated bud revealed by tannic acid-containing fixatives was the morphological entity associated with this intercompartment Golgi apparatus transfer. This report summarizes information from the author's laboratories that suggests that perhaps the majority of these coated buds, while associated with the Golgi apparatus, are not vesicles per se but rather coated ends of tubules. Golgi apparatus tubules have been postulated to permit interconnections among adjacent Golgi apparatus stacks but not to function in transport between contiguous cisternae of the same Golgi apparatus stack.In the interest of scientific discourse, reasoned and constructive replies to views expressed under New Ideas in Cell Biology will be considered for publication. In this case, the responsible editor, to be contacted by respondents, is E. Schnepf.     

The transmembrane domain of murine alpha-mannosidase IB is a major determinant of Golgi localization

Murine alpha1,2-mannosidase IB is a type II transmembrane protein localized to the Golgi apparatus where it is involved in the biogenesis of complex and hybrid N-glycans. This enzyme consists of a cytoplasmic tail, a transmembrane domain followed by a "stem" region and a large C-terminal catalytic domain. To analyze the determinants of targeting, we constructed various deletion mutants of murine alpha1,2-mannosidase IB as well as alpha1,2-mannosidase IB/yeast alpha1,2-mannosidase and alpha1,2-mannosidase IB/GFP chimeras and localized these proteins by fluorescence microscopy, when expressed transiently in COS7 cells. Replacing the catalytic domain of alpha1,2-mannosidase IB with that of the homologous yeast alpha1,2-mannosidase and deleting the "stem" region in this chimera had no effect on Golgi targeting, but caused increased cell surface localization. The N-terminal tagged protein lacking a catalytic domain was also localized to the Golgi. In the latter case, when the stem region was partially or completely removed, the protein was found in both the ER and the Golgi. A chimera consisting of the alpha1,2-mannosidase IB N-terminal region (cytoplasmic and transmembrane domains plus 10 amino acids of the "stem" region) and GFP was localized mainly to the Golgi. Deletion of 30 out of 35 amino acids in the cytoplasmic tail had no effect on Golgi localization. A GFP chimera lacking the entire cytoplasmic tail was found in both the ER and the Golgi. These results indicate that the transmembrane domain of alpha1,2-mannosidase IB is a major determinant of Golgi localization.     

Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack

We describe a scheme for the purification of the nonclathrin-coated vesicles that mediate transport of proteins between Golgi cisternae and probably from ER to Golgi. These "Golgi-derived coated vesicles" accumulate when Golgi membranes are incubated with ATP and cytosol in the presence of GTP gamma S, a compound that blocks vesicle fusion. The coated vesicles dissociate from the Golgi cisternae in high salt and can then be purified by employing differential and density gradient centrifugation. Golgi-derived coated vesicles have a putative polypeptide composition that is distinct from both cytosol and Golgi membranes, as well as from that of clathrin-coated vesicles.     

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.     

Mitotic Golgi fragments in HeLa cells and their role in the reassembly pathway

Immunoelectron microscopy and stereology were used to identify and quantitate Golgi fragments in metaphase HeLa cells and to study Golgi reassembly during telophase. On ultrathin frozen sections of metaphase cells, labeling for the Golgi marker protein, galactosyltransferase, was found over multivesicular Golgi clusters and free vesicles that were found mainly in the mitotic spindle region. The density of Golgi cluster membrane varied from cell to cell and was inversely related to the density of free vesicles in the spindle. There were thousands of free Golgi vesicles and they comprised a significant proportion of the total Golgi membrane. During telophase, the distribution of galactosyltransferase labeling shifted from free Golgi vesicles towards Golgi clusters and the population of free vesicles was depleted. The number of clusters was no more than in metaphase cells so the observed fourfold increase in membrane surface meant that individual clusters had increased in size. More than half of these had cisterna(e) and were located next to "buds" on the endoplasmic reticulum. Early in G1 the number of clusters dropped as they congregated in the juxtanuclear region and fused. These results show that fragmentation of the Golgi apparatus yields Golgi clusters and free vesicles and reassembly from these fragments is at least a two-step process: (a) growth of a limited number of dispersed clusters by accretion and fusion of vesicles to form cisternal clusters next to membranous "buds" on the endoplasmic reticulum; (b) congregation and fusion to form the interphase Golgi stack in the juxtanuclear region.     

Sites of sulfatation in the chondrocytes of the articular cartilage of the rabbit

35S sulfate uptake by the articular cartilage chondrocytes, from biopsies of rabbit, have been studied by high resolution autoradiography. The Golgi apparatus, rough endoplasmic reticulum, cytosol, cytoplasmic membrane and extracellular space were considered as cell compartments in the quantitative analysis of the autoradiograms. The results obtained show: 1) a high activity of radiosotope incorporation in the Golgi apparatus; 2) a fast rhythm of transfer of the substances labelled in the Golgi apparatus to the cell membrane; 3) significant labelling of the rough endoplasmic reticulum, throughout the experiment. It is concluded: 1) The grains observed in the rough endoplasmic reticulum show a significant radioisotope uptake on this level, and this evidence some sulfotransferase activity. 2) The high 35S sulfate uptake level which is observed in the Golgi apparatus demonstrates that the highest sulfotransferase enzyme activity is located in this cell area, thus showing that the "early" sulfation that began in the rough endoplasmic reticulum was completed by a "late" sulfation in the Golgi apparatus. It is here that complete chondromucoprotein building takes place before being excreted. 3) The high transfer level of the labelled substances from the Golgi apparatus shows that the sulfated product secretion for building the cartilage matrix takes place rapidly since a great label increase can be already observed at the beginning of the chase period in the outer surrounding area of the chondrocyte membrane.     

Requirement of the SH4 and tyrosine-kinase domains but not the kinase activity of Lyn for its biosynthetic targeting to caveolin-positive Golgi membranes

Background

The Src-family non-receptor-type tyrosine kinase Lyn, which is often associated with chemotherapeutic resistance in cancer, localizes not only to the plasma membrane but also Golgi membranes. Recently, we showed that Lyn, which is synthesized in the cytosol, is transported from the Golgi to the plasma membrane along the secretory pathway. However, it is still unclear how Golgi targeting of newly synthesized Lyn is regulated.

Methods

Subcellular localization of Lyn and its mutants was determined by confocal microscopy.

Results

We show that the kinase domain, but not the SH3 and SH2 domains, of Lyn is required for the targeting of Lyn to the Golgi, whereas the N-terminal lipids of the Lyn SH4 domain are not sufficient for its Golgi targeting. Although intact Lyn, which colocalizes with caveolin-positive Golgi membranes, can traffic toward the plasma membrane, kinase domain-deleted Lyn is immobilized on caveolin-negative Golgi membranes.

General significance

Besides the SH4 domain, the Lyn kinase domain is important for targeting of newly synthesized Lyn to the Golgi, especially caveolin-positive transport membranes. Our results provide a novel role of the Lyn catalytic domain in the Golgi targeting of newly synthesized Lyn in a manner independent of its kinase activity.     

Golgi complex fragmentation in G2/M transition: An organelle-based cell-cycle checkpoint

In mammalian cells, the Golgi complex is organized into a continuous membranous system known as the Golgi ribbon, which is formed by individual Golgi stacks that are laterally connected by tubular bridges. During mitosis, the Golgi ribbon undergoes extensive fragmentation through a multistage process that is required for its correct partitioning into the daughter cells. Importantly, inhibition of this Golgi disassembly results in cell-cycle arrest at the G2 stage, suggesting that accurate inheritance of the Golgi complex is monitored by a "Golgi mitotic checkpoint." Here, we discuss the mechanisms and regulation of the Golgi ribbon breakdown and briefly comment on how Golgi partitioning may inhibit G2/M transition.     

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).     

A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack

Isolated Golgi membranes incubated in the presence of ATP and a cytosolic protein fraction form a population of coated buds or vesicles from the Golgi cisternae. The coats do not have the characteristic hexagonal-pentagonal basketwork of clathrin, and do not react with anti-clathrin polyclonal antibody. The conditions that produce these apparently nonclathrin-coated buds also reconstitute protein transport between compartments of the Golgi stack. The membrane of the buds contains the glycoprotein in transit through these Golgi stacks (VSV-encoded G protein). This suggests that protein transport through the Golgi stack is mediated by a new type of coated vesicle that does not contain clathrin. The concentration of G protein in the coated buds reflects the local concentration of G protein in the cisternae, raising the possibility that the Golgi coated vesicles may be "bulk" membrane carriers.     

Architecture of the Mammalian Golgi

Since its first visualization in 1898, the Golgi has been a topic of intense morphological research. A typical mammalian Golgi consists of a pile of stapled cisternae, the Golgi stack, which is a key station for modification of newly synthesized proteins and lipids. Distinct stacks are interconnected by tubules to form the Golgi ribbon. At the entrance site of the Golgi, the cis-Golgi, vesicular tubular clusters (VTCs) form the intermediate between the endoplasmic reticulum and the Golgi stack. At the exit site of the Golgi, the trans-Golgi, the trans-Golgi network (TGN) is the major site of sorting proteins to distinct cellular locations. Golgi functioning can only be understood in light of its complex architecture, as was revealed by a range of distinct electron microscopy (EM) approaches. In this article, a general concept of mammalian Golgi architecture, including VTCs and the TGN, is described.In 1898 Camillo Golgi was the first to visualize, describe, and ultimately name the Golgi complex. Using a histochemical impregnation method causing the reduction and deposition of silver, he defined the Golgi in neuronal cells as a reticular apparatus stained by the “black reaction” (Golgi 1898). In the 1950s, the first ultrastructural images of the Golgi were revealed using the then newly developed electron microscope (EM) (Dalton 1954; Farquhar and Rinehart 1954; Sjostrand and Hanzon 1954; Dalton and Felix 1956), reviewed by Farquhar and Palade (1981). In 1961, the thiamine pyrophosphatase reaction developed by Novikoff and Goldfischer allowed cytochemical labeling of Golgi membranes, which revealed the ubiquitous cellular distribution of this organelle (Novikoff and Goldfischer 1961). In the many years of ultrastructural research that have followed, the visualization of the Golgi has gone hand-in-hand with the developing EM techniques.The intriguing structural complexity of the Golgi has made it one of the most photographed organelles in the cell. However, a full understanding of Golgi architecture is hard to deduce from the ultrathin (70–100 nm) sections used in standard transmission EM preparations. Rambourg and Clermont (1974) were the first to investigate the Golgi in three dimensions (3D), using stereoscopy (Rambourg 1974). In this approach a “thick” (150–200 nm), EM section is photographed at two distinct angles, after which the pairs of photographs are viewed with a stereoscope. Over the years, stereoscopy was applied to a variety of cells and has greatly contributed to our current understanding of Golgi architecture (Lindsey and Ellisman 1985; Rambourg and Clermont 1990; Clermont et al. 1994; Clermont et al. 1995). An alternative approach to study 3D structure is serial sectioning, by which a series of adjacent (serial) thin sections are collected. The Golgi can be followed throughout these sections and be constructed into a 3D model (Beams and Kessel 1968; Dylewski et al. 1984; Rambourg and Clermont 1990). In the nineties, 3D-EM was boosted by the introduction of high-voltage, dual axis 3D electron tomography (Ladinsky et al. 1999; Koster and Klumperman 2003; Marsh 2005; Marsh 2007; Noske et al. 2008), which allows the analysis of sections of up to 3–4 µm with a 4–6 nm resolution in the z-axis. The sections are photographed in a tilt series of different angles, which are reconstructed into a 3D tomogram that allows one to “look beyond” a given structure and reveals how it relates to other cellular compartments.Membranes with a similar appearance can differ in protein content and function. These differences are revealed by protein localization techniques. Therefore, in addition to the “classical” EM techniques providing ultrastructural details, EM methods that determine protein localization within the context of the cellular morphology have been crucial to further our understanding on the functional organization of the Golgi. For example, by enzyme-activity-based cytochemical staining the cis-to-trans-polarity in the distribution of Golgi glycosylation enzymes was discovered, reviewed by Farquhar and Palade (1981), which was key to understanding the functional organization of the Golgi stack in protein and lipid glycosylation. With the development of immunoEM methods, using antibodies, the need for enzyme activity for protein localization was overcome. This paved the way for the localization of a wide variety of proteins, such as the cytoplasmic coat complexes associated with the Golgi (Rabouille and Klumperman 2005).A logical next step in EM-based imaging of the Golgi would be to combine protein localization with 3D imaging, but this is technically challenging. A number of protocols enabling protein localization in 3D have recently been described (Trucco et al. 2004; Grabenbauer et al. 2005; Gaietta et al. 2006; Zeuschner et al. 2006; Meiblitzer-Ruppitsch et al. 2008), but these have only been applied in a limited manner to Golgi studies. Another approach that holds great potential for Golgi research is correlative microscopy (CLEM). Live cell imaging of fluorescent proteins has revolutionized cell biology by the real time visualization of dynamic events. However, live cell imaging does not reveal membrane complexity. By CLEM, live cells are first viewed by light microscopy and then prepared for EM (Mironov et al. 2008; van Rijnsoever et al. 2008). When coupled with the recent introduction of super resolution light microscopy techniques for real time imaging, the combination with EM for direct correlation with ultrastructural resolution has great potential (Hell 2009; Lippincott-Schwartz and Manley 2009).The 100th anniversary of the discovery of the Golgi, in 1998, triggered a wave of reviews on this organelle, including those focusing on Golgi architecture (Rambourg 1997; Farquhar and Palade 1998). More recent reviews that describe Golgi structure in great detail are provided by Marsh (2005) and Hua (2009). In this article, the most recent insights in mammalian Golgi architecture as revealed by distinct EM approaches are integrated into a general concept.     

The Golgi apparatus in the acinar cells of the developing embryonic pancreas: I. Morphology and enzyme cytochemistry

The Golgi apparatus of pancreatic acinar cells of rat embryos was studied during development from day 13 through day 20 of gestation. The morphological and enzyme cytochemical patterns varied characteristically in the course of cell differentiation. A pronounced system of "rigid lamellae" characterized the area near the trans face of the Golgi stacks in the protodifferentiated and early phases of the differentiated states; by contrast, "rigid lamellae" were sparse in the terminal period of gestation. Reaction product of acid phosphatase labeled the "rigid lamellae" in the protodifferentiated state, was extended across the majority of the stacked cisternae in the early differentiated state, but was restricted to the trans side again in the later periods of cell differentiation. The early phase of the differentiated state was characterized by the tight association of the endoplasmic reticulum and Golgi cisternae on the trans side; the close spatial relationship of the two compartments was lessened after production of secretion granules had started. The findings are in line with those of recent studies on the Golgi organization in some other types of cells in different functional states, and they present the embryonic pancreatic tissue as another model for demonstrating the high flexibility of the Golgi complex. In agreement with the patterns previously found in the absorptive cells of the small intestine, the present results show that the close associations of the endoplasmic reticulum and cisternae of the trans Golgi side predominate in the early stages of cell differentiation.     

Involvement of GTP-binding "G" proteins in transport through the Golgi stack

GTP gamma S irreversibly inhibits protein transport between successive compartments of the Golgi stack in a cell-free system. Fluoride, potentiated by the addition of aluminum ion, also causes a strong inhibition. These are hallmarks of the involvement of a guanine nucleotide-binding or regulatory "G" protein. Inhibition by GTP gamma S requires a cytosolic inhibitory factor that binds to Golgi membranes during inhibition. Preincubation experiments reveal that GTP gamma S blocks the function of acceptor Golgi but not donor Golgi membranes. More specifically, a processing step in between vesicle attachment and the actual fusion event seems to be affected. Electron microscopy demonstrates a corresponding 5-fold accumulation of non-clathrin-coated buds and vesicles associated with the Golgi cisternae during inhibition by GTP gamma S.     

Regional differences in the distribution of golgi cells in the cerebellar cortex of man and some other mammals

Summary The number of Golgi cells per unit volume was determined in different regions of the cerebellar cortex of man and of ten other mammals. Despite the general belief in the uniform architecture of the cerebellar cortex, regional differences in the distribution of Golgi cells were found. In the inferior parts of the vermis, the number of Golgi cells per unit volume is twice that in the corresponding hemispheres. In addition, there are differences between the anterior and inferior parts of the vermis. These differences are a feature of the cytoarchitecture of the cerebellum in man and all the investigated mammals. The ratio of Purkinje cells to Golgi cells was also determined and found to differ in different species. In man, this ratio is 11.5, while in the monkey and cat it is almost 11.9 and in the rat 13.3. These differences in the ratio of Purkinje cells to Golgi cells are discussed from the point of view of cerebellar evolution.Supported by the Deutsche Forschungsgemeinschaft.     

GOLPH3 keeps the Golgi residents at home

In this issue of JCB, Welch et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202106115) show that GOLPH3 mediates the sorting of numerous Golgi proteins into recycling COPI transport vesicles. This explains how many resident proteins are retained at the Golgi and reveals a key role for GOLPH3 in maintaining Golgi homeostasis.

The Golgi apparatus lies at the heart of the secretory pathway, where its major functions are the posttranslational modification of cargo proteins and lipids, particularly at the level of glycosylation, and the sorting of cargo to its correct onward destination. The Golgi is composed of stacked membrane compartments called cisternae, which contain numerous resident enzymes that act on the cargo as it passes through the organelle, from the entry or cis side to the exit or trans side. Each resident enzyme has its own distribution within the Golgi stack, resulting in the sequential modification of the secretory cargo as it moves through the Golgi.Various mechanisms exist to ensure that Golgi residents are retained within the Golgi despite the huge flux of protein and lipid through this organelle (1). Major players are COPI vesicles, which recycle Golgi residents from later to earlier cisternae, at the same time as the cisternae are thought to slowly migrate across the stack, as on a conveyor belt, progressively changing composition in a process referred to as cisternal maturation (2). Unlike the Golgi resident enzymes, which enter recycling vesicles, cargo is thought to remain within the maturing cisternae as it moves through the Golgi. Certain Golgi enzymes can bind directly to the COPI coat, explaining their inclusion in COPI vesicles (3), but for other enzymes and resident proteins, their retention mechanism is less obvious.Previous studies on the peripheral Golgi membrane protein GOLPH3 and its paralogue GOLPH3L (herein I will refer to both proteins as GOLPH3) indicated it can bind to certain Golgi enzymes and to the COPI coat, thereby acting as an adaptor to mediate sorting of these enzymes into COPI vesicles (4, 5). This was first shown for the yeast orthologue Vps74p (6, 7) and has also been demonstrated for the Drosophila version of the protein (8), consistent with a conserved function in Golgi enzyme retention. However, the extent to which GOLPH3 might participate in retention of different Golgi enzymes and other resident proteins, and its importance relative to other methods of protein retention in the Golgi, has remained unclear. Indeed, a recent study suggested that GOLPH3 selectively mediates the retention of enzymes involved in glycosphingolipid synthesis, consistent with a fairly selective role in retaining only a subset of resident Golgi enzymes (9). It should also be noted that GOLPH3 has been implicated in other functions, namely budding of exocytic vesicles from the Golgi, the DNA damage response, and mechanistic target of rapamycin signaling (10).In their current paper, Welch et al. used a combination of approaches to reassess the role of GOLPH3 at the Golgi (11). Using proteomics, they could identify numerous GOLPH3 binding partners, which included COPI, as expected, and a large number of other Golgi residents, including numerous Golgi enzymes and other membrane proteins. The ability of GOLPH3 to retain enzymes at the Golgi was confirmed using microscopy and an innovative flow cytometry–based assay to quantify surface versus Golgi abundance. The large number of possible interactors suggested that GOLPH3 could mediate the Golgi retention of many proteins. To further assess this possibility, the authors took advantage of previous observations showing that Golgi enzymes may be misrouted to the lysosome and degraded upon their failure to be retained in the Golgi (6, 7, 9). Using mass spectrometry, they could show that numerous Golgi resident proteins were depleted in GOLPH3 knockout cells, many of which were also found in the GOLPH3 interactome. This included many enzymes involved in glycosylation, consistent with GOLPH3 playing an important role in maintaining Golgi-dependent glycosylation of proteins and lipids. This was supported by lectin analysis, which showed marked changes in a broad range of glycans in the GOLPH3 knockout cells.The large number of GOLPH3 clients raises the question as to how it can recognize so many proteins. Previous work has shown binding to the cytoplasmic tails of Golgi enzymes and an interaction motif has been described for Vps74p and more recently for GOLPH3 (6, 9). However, bioinformatics analysis of the many GOLPH3 clients combined with mutational analysis, as performed in the current study, revealed the lack of a consensus sequence for GOLPH3 binding, with the common feature being a strong net positive charge combined with short cytoplasmic tail length. This would result in a high positive charge proximal to the membrane, which likely allows interaction with an acidic patch on the surface of GOLPH3. This mode of binding could mediate selective retention of many Golgi residents, while allowing for the forward trafficking of cargo proteins that have longer, less charged, or folded cytoplasmic domains.GOLPH3 is an oncogene associated with many types of cancer (12). Several mechanisms have been proposed to account for the oncogenic properties of GOLPH3, but most compelling is that changes in glycosylation are responsible. It was recently shown that GOLPH3-dependent changes in glycosphingolipids affects cell growth by altering mitogenic signaling (9). Changes in glycosylation of surface receptors has also been reported, which can affect surface abundance and hence signaling (13). The new results from Welch et al. suggest that glycosylation of many proteins and lipids may be relevant in cancer and that potentially a broad range of downstream targets contribute to oncogenesis. Such targets could influence processes beyond signaling, including cell adhesion and migration, that are known to be sensitive to changes in the surface glycome and which have been reported in previous studies on GOLPH3 (12).The study by Welch et al. indicates a major role for GOLPH3 in Golgi protein retention (Fig. 1). Clearly though, other retention mechanisms exist, including direct binding to COPI, and transmembrane domain length is also important, where the short transmembrane domain of resident proteins favors partitioning into recycling COPI vesicles and Golgi cisternal membranes of a similar thickness (1). Additional COPI adaptors are also likely, with TM9SF2 recently identified as a likely candidate, being present in Golgi vesicles and able to bind certain Golgi enzymes (1). It is possible that different resident proteins use different adaptors, or that a combination of retention mechanisms act in conjunction for certain residents, providing robustness to the retention process. However, any redundancy would seem incomplete given the strong phenotype seen upon loss of GOLPH3. GOLPH3 is localized toward the trans side of the Golgi, so it is possible that other adaptors, such as TM9SF2 and possibly others, might act earlier in the Golgi, or that direct coat binding is more important within the early Golgi. Hence different residents may be more likely to use different retention mechanisms depending on their location in the Golgi. Because GOLPH3 acts late in the Golgi and can bind many clients, we may think of it as a gatekeeper to prevent loss of numerous Golgi residents from the organelle.Open in a separate windowFigure 1.GOLPH3 plays a major role in Golgi protein retention. Golgi resident proteins, including many glycosylation enzymes, depicted by lollipops, are sorted into recycling COPI vesicles to maintain retention in the Golgi in the face of onward cisternal maturation and secretory cargo transport. Different enzymes are depicted by different lollipop shapes and colors, with GOLPH3 clients indicated by horizontal ovals. Enzymes retained by other mechanisms are depicted by lollipops with circles (transmembrane domain length), squares or vertical ovals (binding to other COPI adaptors, indicated in turquoise and purple), or hexagons (direct binding to the COPI coat). GOLPH3, which is more abundant toward the trans side of the Golgi, has many clients.With regard to possible future studies, although we have a good idea of how GOLPH3 recognizes its clients, detailed structural analysis will prove informative in elucidating how it can bind so many proteins. Similarly, identification of additional adaptors linking Golgi residents to the COPI coat will be important to generate a more comprehensive view of Golgi protein retention. Finally, in the context of disease, further analysis of the glycoproteins and glycolipids whose levels are altered because of changes in GOLPH3 expression, of which there are likely to be many, should provide significant new insights into the mechanisms underlying GOLPH3-mediated tumorigenesis.     

Effect of monensin on the Golgi apparatus of absorptive cells in the small intestine of the rat

Summary The effect of short-time treatment with the ionophore monensin, administered intraluminally at concentrations of 5 and 10 M, was studied on the Golgi apparatus of absorptive cells in the small intestine of the rat. At 2–3 min after treatment most of the Golgi stacks exhibited dilated cisternae. At 4–5 min stacked cisternae were absent; they were replaced by groups of smooth-surfaced vacuoles. Dilatation and vacuolization occurred in the entire stacks without preferential effect on any particular Golgi subcompartment.Monensin did not influence the cytochemical Golgi reaction of thiamine pyrophosphatase and acid phosphatase. The characteristic staining pattern of these two enzymes in all Golgi cisternae of absorptive cells in the proximal small intestine, and the reactivity restricted to trans cisternae in distal segments of the small intestine, were unchanged after treatment with monensin. In the distal small intestine, the cytochemical pattern allowed the monensin-induced vacuoles to be attributed to the former cisor trans-Golgi face. Further, the cytochemical results demonstrate that vacuolization is not restricted to the stacked cisternae, but includes the trans-most cisterna. The latter, usually located at some distance from the Golgi stacks, has been defined as belonging to the GERL system in several types of cells. The clear response to monensin, an agent that selectively affects the Golgi apparatus, indicates common properties between trans-most and stacked Golgi cisternae.     

Identification and Characterization of a Golgi-Localized UDP-Xylose Transporter Family from Arabidopsis

Most glycosylation reactions require activated glycosyl donors in the form of nucleotide sugars to drive processes such as posttranslational modifications and polysaccharide biosynthesis. Most plant cell wall polysaccharides are biosynthesized in the Golgi apparatus from cytosolic-derived nucleotide sugars, which are actively transferred into the Golgi lumen by nucleotide sugar transporters (NSTs). An exception is UDP-xylose, which is biosynthesized in both the cytosol and the Golgi lumen by a family of UDP-xylose synthases. The NST-based transport of UDP-xylose into the Golgi lumen would appear to be redundant. However, employing a recently developed approach, we identified three UDP-xylose transporters in the Arabidopsis thaliana NST family and designated them UDP-XYLOSE TRANSPORTER1 (UXT1) to UXT3. All three transporters localize to the Golgi apparatus, and UXT1 also localizes to the endoplasmic reticulum. Mutants in UXT1 exhibit ∼30% reduction in xylose in stem cell walls. These findings support the importance of the cytosolic UDP-xylose pool and UDP-xylose transporters in cell wall biosynthesis.