A century of Golgi

The Golgi Apparatus

edited by E. G. Berger and J. Roth, Birkhäuser Verlag, 1997. £64.00 (306 pages) ISBN 3 7643 5692 8     

Sphingomyelin organization is required for vesicle biogenesis at the Golgi complex

Sphingomyelin and cholesterol can assemble into domains and segregate from other lipids in the membranes. These domains are reported to function as platforms for protein transport and signalling. Do similar domains exist in the Golgi membranes and are they required for protein secretion? We tested this hypothesis by using D ‐ceramide‐C6 to manipulate lipid homeostasis of the Golgi membranes. Lipidomics of the Golgi membranes isolated from D ‐ceramide‐C6‐treated HeLa cells revealed an increase in the levels of C6‐sphingomyelin, C6‐glucosylceramide, and diacylglycerol. D ‐ceramide‐C6 treatment in HeLa cells inhibited transport carrier formation at the Golgi membranes without affecting the fusion of incoming carriers. The defect in protein secretion as a result of D ‐ceramide‐C6 treatment was alleviated by knockdown of the sphingomyelin synthases 1 and 2. C6‐sphingomyelin prevented liquid‐ordered domain formation in giant unilamellar vesicles and reduced the lipid order in the Golgi membranes of HeLa cells. These findings highlight the importance of a regulated production and organization of sphingomyelin in the biogenesis of transport carriers at the Golgi membranes.     

Fusogenic domains of golgi membranes are sequestered into specialized regions of the stack that can be released by mechanical fragmentation.

A well-characterized cell-free assay that reconstitutes Golgi transport is shown to require physically fragmented Golgi fractions for maximal activity. A Golgi fraction containing large, highly stacked flattened cisternae associated with coatomer-rich components was inactive in the intra-Golgi transport assay. In contrast, more fragmented hepatic Golgi fractions of lower purity were highly active in this assay. Control experiments ruled out defects in glycosylation, the presence of excess coatomer or inhibitory factors, as well as the lack or consumption of limiting diffusible factors as responsible for the lower activity of intact Golgi fractions. Neither Brefeldin A treatment, preincubation with KCl (that completely removed associated coatomer) or preincubation with imidazole buffers that caused unstacking, activated stacked fractions for transport. Only physical fragmentation promoted recovery of Golgi fractions active for transport in vitro. Rate-zonal centrifugation partially separated smaller transport-active Golgi fragments with a unique v-SNARE pattern, away from the bulk of Golgi-derived elements identified by their morphology and content of Golgi marker enzymes (N-acetyl glucosaminyl and galactosyl transferase activities). These fragments released during activation likely represent intra-Golgi continuities involved in maintaining the dynamic redistribution of resident enzymes during rapid anterograde transport of secretory cargo through the Golgi in vivo.     

Lead staining in the Golgi complex

Lead ions at similar concentrations to those used for Gomori type phosphatase localization stain some parts of the vacuolar system, particularly compartments of the Golgi complex (GC) and isolation envelopes (im) in a characteristic way in both vertebrates and invertebrates. After fixation in 2.5% glutaraldehyde, lead citrate in acetate or aspartate buffer (pH 5.5-7.2) leaves the contents of GC cisternal compartments with a fine particulate stippling. In the fat body of Calpodes ethlius and in mouse pancreas the staining is faint but definite without further enhancement of contrast, although it is easily overlooked after section staining. The distribution of lead stain differs from that of the lead phosphate precipitated after Gomori type acid phosphatase reactions. Whereas lead stain may be in all GC and im compartments, acid phosphatase is restricted to the innermost saccules and nearby vacuoles. The compartment specific staining by led also differs from the generalized staining in all compartments given by uranyl. Thus the contents of luminal membrane surfaces of some parts of the vacuolar system can be characterized by their ability to bind lead. In cells where protein synthesis has been blocked by cycloheximide, secretory vesicles are absent and the RER and GC from the generalized staining in all compartments given by uranyl. Thus the contents of luminal membrane surfaces of some parts of the vacuolar system can be characterized by their ability to bind lead. In cells where protein synthesis has been blocked by cycloheximide, secretory vesicles are absent and the RER and GC from the generalized staining in all compartments given by uranyl. Thus the contents of luminal membrane surfaces of some parts of the vacuolar system can be characterized by their ability to bind lead. In cells where protein synthesis has been blocked by cycloheximide, secretory vesicles are absent and the RER and GC cisternae are devoid of uranyl stainable material. However, lead staining and acid phosphatase activity in the GC continue. We presume that they mark the environment within these cisternae rather than the proteins passing through them. This environment is itself not static. Several observations suggest that the function of cisternae that is detectable by lead staining is temporally discontinuous and related to a stage of maturation or development. Only early stage ims stain: the staining ceases by the beginning of autophagy after hydrolytic enzymes are presumed to have been added. Condensing vacuoles cease to stain as the central core crystallizes out. Stain may be absent from one or two GC saccules at any position in the stack as though the phase of lead staining (or lack or it) can move progressively through the system. We conclude that in studies characterizing components of the vacuolar system it is necessary to separate those that mark transient occupants of a compartment from those that mark the compartment itself. Both may vary temporally independently from one another.     

ER/Golgi intermediates acquire Golgi enzymes by brefeldin A-sensitive retrograde transport in vitro

Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I-mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.     

Direct interaction between the COG complex and the SM protein,Sly1, is required for Golgi SNARE pairing

The crucial roles of Sec1/Munc18 (SM)‐like proteins in membrane fusion have been evidenced in genetic and biochemical studies. SM proteins interact directly with SNAREs and contribute to SNARE pairing by a yet unclear mechanism. Here, we show that the SM protein, Sly1, interacts directly with the conserved oligomeric Golgi (COG) tethering complex. The Sly1–COG interaction is mediated by the Cog4 subunit, which also interacts with Syntaxin 5 through a different binding site. We provide evidence that disruption of Cog4–Sly1 interaction impairs pairing of SNAREs involved in intra‐Golgi transport thereby markedly attenuating Golgi‐to‐ER retrograde transport. These results highlight the mechanism by which SM proteins link tethering to SNAREpin assembly.     

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus

Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune‐dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species‐dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA‐dependent secretory pathway. This led to a general inhibition of protein secretion by PDT‐treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin‐based PDT. Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro‐apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function.     

Sorting out glycosylation enzymes in the Golgi apparatus

The study of glycosylation and glycosylation enzymes has been instrumental for the advancement of Cell Biology. After Neutra and Leblond showed that the Golgi apparatus is the main site of glycosylation, elucidation of oligosaccharide structures by Baenziger and Kornfeld and subsequent mapping of glycosylation enzymes followed. This enabled development of an in vitro transport assay by Rothman and co-workers using glycosylation to monitor intra Golgi transport which, complemented by yeast genetics by Schekman and co-workers, provided much of the fundamental insights and key components of the secretory pathway that we today take for granted. Glycobiology continues to play a key role in Cell Biology and here, we look at the use of glycosylation enzymes to elucidate intra Golgi transport.     

高尔基体对小鼠卵母细胞体外发育进程的影响

目的初步探讨高尔基体在小鼠卵母细胞体外发育进程中的作用。方法布雷菲德菌素A（Brefeldin A,BFA）处理小鼠未成熟,成熟卵母细胞,利用特异性标记物阻COP标记高尔基体。激光扫描共聚焦显微镜观察BFA处理对高尔基体产生的影响;同时。观察并比较不同处理组小鼠未成熟／成熟卵母细胞的体外成熟率、孤雌激活率、体外受精率及2-细胞率。结果GV期卵母细胞经BFA处理后,高尔基体的形态和分布发生明显改变。其体外成熟率（2．5％）与对照组（70．4％）比较统计学差异显著（P〈0．001）;洗掉BFA后,其体外成熟率（67．2％）与对照组无统计学差异（P〉0．05）。另外,成熟卵母细胞经BFA处理后。其体外受精率及2．细胞率均与对照组差异无统计学意义（P〉0．05）。结论小鼠卵母细胞体外成熟的正常进行需要高尔基体主导的膜运输。而体外受精和受精卵卵裂过程中不需要功能性的高尔基体。     

ER to Golgi transport: Requirement for p115 at a pre-Golgi VTC stage

The membrane transport factor p115 functions in the secretory pathway of mammalian cells. Using biochemical and morphological approaches, we show that p115 participates in the assembly and maintenance of normal Golgi structure and is required for ER to Golgi traffic at a pre-Golgi stage. Injection of antibodies against p115 into intact WIF-B cells caused Golgi disruption and inhibited Golgi complex reassembly after BFA treatment and wash-out. Addition of anti-p115 antibodies or depletion of p115 from a VSVtsO45 based semi-intact cell transport assay inhibited transport. The inhibition occurred after VSV glycoprotein (VSV-G) exit from the ER but before its delivery to the Golgi complex, and resulted in VSV-G protein accumulating in peripheral vesicular tubular clusters (VTCs). The p115-requiring step of transport followed the rab1-requiring step and preceded the Ca(2+)-requiring step. Unexpectedly, mannosidase I redistributed from the Golgi complex to colocalize with VSV-G protein arrested in pre-Golgi VTCs by p115 depletion. Redistribution of mannosidase I was also observed in cells incubated at 15 degrees C. Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack. This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.     

Proteoglycan Synthesis and Golgi Organization in Polarized Epithelial Cells

A large number of complex glycosylation mechanisms take place in the Golgi apparatus. In epithelial cells, glycosylated protein molecules are transported to both the apical and the basolateral surface domains. Although the prevailing view is that the Golgi apparatus provides the same lumenal environment for glycosylation of apical and basolateral cargo proteins, there are indications that proteoglycans destined for the two opposite epithelial surfaces are exposed to different conditions in transit through the Golgi apparatus. We will here review data relating proteoglycan and glycoprotein synthesis to characteristics of the apical and basolateral secretory pathways in epithelial cells.     

Reconstitution of Retrograde Transport from the Golgi to the ER In Vitro

Retrograde transport from the Golgi to the ER is an essential process. Resident ER proteins that escape the ER and proteins that cycle between the Golgi and the ER must be retrieved. The interdependence of anterograde and retrograde vesicle trafficking makes the dissection of both processes difficult in vivo. We have developed an in vitro system that measures the retrieval of a soluble reporter protein, the precursor of the yeast pheromone α-factor fused to a retrieval signal (HDEL) at its COOH terminus (Dean, N., and H.R.B Pelham. 1990. J. Cell Biol. 111:369–377). Retrieval depends on the HDEL sequence; the α-factor precursor, naturally lacking this sequence, is not retrieved. A full cycle of anterograde and retrograde transport requires a simple set of purified cytosolic proteins, including Sec18p, the Lma1p complex, Uso1p, coatomer, and Arf1p. Among the membrane-bound v-SNAP receptor (v-SNARE) proteins, Bos1p is required only for forward transport, Sec22p only for retrograde trafficking, and Bet1p is implicated in both avenues of transport. Putative retrograde carriers (COPI vesicles) generated from Golgi-enriched membranes contain v-SNAREs as well as Emp47p as cargo.     

The arrangement of the Golgi complex beads is not controlled by the cytoskeleton

Exposure of insect fat body to treatments which disrupt microtubules (colchicine, vinblastine sulfate and cold treatment) blocks intracellular transport between the Golgi complex and the plasma membrane but does not affect Golgi complex bead rings or transport from rough endoplasmic reticulum to the Golgi complex. Drugs which disrupt microfilaments (cytochalasins B and D) do not affect the bead rings or intracellular transport of secretory proteins at any level. Thus, intracellular transport between the rough endoplasmic reticulum and the Golgi complex and the arrangement of the beads in rings are both independent of the cytoskeleton. The ring arrangement is presumably maintained by interconnection(s) with rough endoplasmic reticulum membrane.     

Effect of cadmium on Golgi complex of freshwater teleost (Pimelodus maculatus) hepatocytes

Summary Freshwater teleost hepatocytes show hypertrophied Golgi complexes 3 days after a single intraperitoneal injection of cadmium chloride (10 mg/Kg).     

Evidence for regulation of ER/Golgi SNARE complex formation by hsc70 chaperones

SNARE proteins control intracellular membrane fusion through formation of membrane-bridging helix bundles of amphipathic SNARE motifs. Repetitive cycles of membrane fusion likely involve repetitive folding/unfolding of the SNARE motif helical structure. Despite these conformational demands, little is known about conformational regulation of SNAREs by other proteins. Here we demonstrate that hsc70 chaperones stimulate in vitro SNARE complex formation among the ER/Golgi SNAREs syntaxin 5, membrin, rbetl and sec22b, under conditions in which assembly is normally inhibited. Thus, molecular chaperones can render the SNARE motif more competent for assembly. Partially purified hsc70 fractions from brain cytosol had higher specific activities than fully purified hsc70, suggesting the involvement of unidentified cofactors. Using chemical crosslinking of cells followed by immunoprecipitation, we found that hsc70 was associated with ER/Golgi SNAREs in vivo. Consistent with a modulatory role for hsc70 in transport, we found that excess hsc70 specifically inhibited ER-to-Golgi transport in permeabilized cells.