Molecular basis for Golgi maintenance and biogenesis

The Golgi apparatus contains thousands of different types of integral and peripheral membrane proteins, perhaps more than any other intracellular organelle. To understand these proteins' roles in Golgi function and in broader cellular processes, it is useful to categorize them according to their contribution to Golgi creation and maintenance. This is because all of the Golgi's functions derive from its ability to maintain steady-state pools of particular proteins and lipids, which in turn relies on the Golgi's dynamic character - that is, its ongoing state of transformation and outgrowth from the endoplasmic reticulum. Here, we categorize the expanding list of Golgi-associated proteins on the basis of their role in Golgi reformation after the Golgi has been disassembled. Information gained on how different proteins participate in this process can provide important insights for understanding the Golgi's global functions within cells.     

Cell cycle maintenance and biogenesis of the Golgi complex

How organelle identity is established and maintained, and how organelles divide and partition between daughter cells, are central questions of organelle biology. For the membrane-bound organelles of the secretory and endocytic pathways [including the endoplasmic reticulum (ER), Golgi complex, lysosomes, and endosomes], answering these questions has proved difficult because these organelles undergo continuous exchange of material. As a result, many resident proteins are not localized to a single site, organelle boundaries overlap, and when interorganellar membrane flow is interrupted, organelle structure is altered. The existence and identity of these organelles, therefore, appears to be a product of the dynamic processes of membrane trafficking and sorting. This is particularly true for the Golgi complex, which resides and functions at the crossroads of the secretory pathway. The Golgi receives newly synthesized proteins from the ER, covalently modifies them, and then distributes them to various final destinations within the cell. In addition, the Golgi recycles selected components back to the ER. These activities result from the Golgi's distinctive membranes, which are organized as polarized stacks (cis to trans) of flattened cisternae surrounded by tubules and vesicles. Golgi membranes are highly dynamic despite their characteristic organization and morphology, undergoing rapid disassembly and reassembly during mitosis and in response to perturbations in membrane trafficking pathways. How Golgi membranes fragment and disperse under these conditions is only beginning to be clarified, but is central to understanding the mechanism(s) underlying Golgi identity and biogenesis. Recent work, discussed in this review, suggests that membrane recycling pathways operating between the Golgi and ER play an indispensable role in Golgi maintenance and biogenesis, with the Golgi dispersing and reforming through the intermediary of the ER both in mitosis and in interphase when membrane cycling pathways are disrupted.     

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.     

Sec7p directs the transitions required for yeast Golgi biogenesis

Endoplasmic reticulum (ER)-to-Golgi traffic in yeast proceeds by the maturation of membrane compartments from post-ER vesicles to intermediate small vesicle tubular clusters (VTCs) to Golgi nodular membrane networks (Morin-Ganet et al., Traffic 2000; 1: 56–68). The balance between ER and Golgi compartments is maintained by COPII- and COPI-mediated anterograde and retrograde traffic, which are dependent on Sec7p and ARF function. The sec7-4 temperature-sensitive allele is a mutation in the highly conserved Sec7 domain (Sec7d) found in all ARF-guanine nucleotide exchange factor proteins. Post-ER trafficking is rapidly inactivated in sec7-4 mutant yeast at the restrictive temperature. This conditional defect prevented the normal production of VTCs and instead generated Golgi-like tubes emanating from the ER exit sites. These tubes progressively developed into stacked cisternae defining the landmark sec7 mutant phenotype. Consistent with the in vivo results, a Sec7d peptide inhibited ER-to-Golgi transport and displaced Sec7p from its membrane anchor in vitro . The similarities in the consequences of inactivating Sec7p or ARFs in vivo was revealed by genetic disruption of yeast ARFs or by addition of brefeldin A (BFA) to whole cells. These treatments, as in sec7-4 yeast, affected the morphology of membrane compartments in the ER-Golgi transition. Further evidence for Sec7p involvement in the transition for Golgi biogenesis was revealed by in vitro binding between distinct domains of Sec7p with ARFs, COPI and COPII coat proteins. These results suggest that Sec7p coordinates membrane transitions in Golgi biogenesis by directing and scaffolding the binding and disassembly of coat protein complexes to membranes, both at the VTC transition from ER exit sites to form Golgi elements and for later events in Golgi maturation.     

p97/p47-Mediated biogenesis of Golgi and ER

In mammalian cells, the Golgi apparatus and endoplasmic reticulum have typical structures during interphase: stacked cisternae located adjacent to the nucleus and a network of interconnected tubules throughout the cytoplasm, respectively. At mitosis their architectures disappear and are reassembled in daughter cells. p97, an AAA-ATPase, mediates membrane fusion and is required for reassembly of these organelles. In the p97-mediated membrane fusion, p47 was identified as an essential cofactor, through which p97 binds to a SNARE, syntaxin5. A second essential cofactor, VCIP135, was identified as a p97/p47/syntaxin5-interacting protein. Several lines of recent evidence suggest that ubiquitination may be implicated in the p97/p47 pathway; p47 binds to monoubiquitinated proteins and VCIP135 shows a deubiquitinating activity in vitro. For the cell-cycle regulation of the p97/p47 pathway, it has been reported that the localization and phosphorylation-dephosphorylation of p47 are crucial. In this review, we describe the components involved in the p97-mediated membrane fusion and discuss the regulation of the fusion pathway.     

Polo-like kinase is required for Golgi and bilobe biogenesis in Trypanosoma brucei

A bilobed structure marked by TbCentrin2 regulates Golgi duplication in the protozoan parasite Trypanosoma brucei. This structure must itself duplicate during the cell cycle for Golgi inheritance to proceed normally. We show here that duplication of the bilobed structure is dependent on the single polo-like kinase (PLK) homologue in T. brucei (TbPLK). Depletion of TbPLK leads to malformed bilobed structures, which is consistent with an inhibition of duplication and an increase in the number of dispersed Golgi structures with associated endoplasmic reticulum exit sites. These data suggest that the bilobe may act as a scaffold for the controlled assembly of the duplicating Golgi.     

Architecture of the Golgi apparatus of a scale-forming alga: biogenesis and transport of scales

Mechanisms of transport of secretory products across the Golgi apparatus (GA) as well as of scale formation in prymnesiophytes have remained controversial. We have used a quantitative morphological approach to study formation and transport of scales across the GA in haploid cells of Pleurochrysis sp. The GA of these cells differs from the GA of higher plants in at least six morphological characteristics. Our results show that scales form in the trans-Golgi network (TGN) and transit the TGN in heretofore unrecognized prosecretory vesicles. Prosecretory vesicles differentiate into secretory vesicles prior to exocytosis of scales to the cell surface. Because prosecretory vesicles are only fragments of TGN cisternae, the classical model of cisternal progression is not a valid mechanism of transport in this alga. TGN transport vesicles are also involved in scale formation; however, the role of tubular connections between cisternae of a single stack-TGN unit is not clear. The relationship of two morphological types of cisternal dilations to a membrane-associated, bottlebrush-shaped macromolecule of novel morphology suggests a new hypothesis for the biogenesis of scales.     

Building a secretory apparatus: role of ARF1/COPI in Golgi biogenesis and maintenance

 The secretory apparatus within all eukaryotic cells comprises a dynamic membrane system with bidirectional membrane transport pathways and overlapping compartmental boundaries. Membrane traffic and organelle biogenesis/maintenance are fundamentally linked within this system, with perturbations in membrane traffic quickly leading to changes in organelle structure and identity. Dissection of the molecular basis of these properties in yeast and mammalian cells has revealed a crucial role for the cytoplasmic protein complex ARF1/COPI, which undergoes regulated assembly and disassembly with membranes. ARF1/COPI appears to be involved in the formation and maintenance of the Golgi complex, which is the receiving and delivery station for all secretory traffic. ARF1-GTP, through assembly of COPI to membranes and, possibly, through activation of PLD, is likely to promote the formation and maturation of pre-Golgi intermediates into Golgi elements, whereas ARF-GDP causes COPI dissociation and stimulates the formation of retrograde transport structures that recycle Golgi membrane back to the ER. These processes are appear to underlie the coupling of organelle biogenesis and membrane trafficking within cells, allowing the size and shape of secretory organelles to be altered in response to changing cellular needs. Future work needs to address how the activation and localization of ARF1/COPI to membranes as well as other related factors are temporally and spatially regulated, and by what mechanism they transform membrane shape and dynamics to facilitate protein transport and compartmental functioning. Accepted: 23 March 1998     

Structure of the membrane-tethering GRASP domain reveals a unique PDZ ligand interaction that mediates Golgi biogenesis

Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.     

Peroxisome biogenesis and peroxisome biogenesis disorders

Peroxisome assembly in mammals requires more than 15 genes. Two isoforms of the peroxisome targeting signal type 1 (PTS1) receptor, Pex5pS and Pex5pL, are identified in mammals. Pex5pS and Pex5pL bind PTS1 proteins. Pex5pL, but not Pex5pS, directly interacts with the PTS2 receptor, Pex7p, carrying its cargo PTS2 protein in the cytosol. Pex5p carrying the cargos, PTS1 and PTS2, docks with the initial site Pex14p in a putative import machinery, subsequently translocating to other components such as Pex13p, Pex2p, Pex10p and Pex12p, whereby the matrix proteins are imported. The peroxins, Pex3p, Pex16p and Pex19p, function in the assembly of peroxisomal membrane vesicles that precedes the import of matrix proteins. Hence, peroxisomes may form de novo and do not have to arise from pre-existing, morphologically recognizable peroxisomes. Impaired peroxisome assembly causes peroxisome biogenesis disorders such as Zellweger syndrome.     

The biogenesis of the Golgi ribbon: the roles of membrane input from the ER and of GM130

The Golgi complex in mammalian cells forms a continuous ribbon of interconnected stacks of flat cisternae. We show here that this distinctive architecture reflects and requires the continuous input of membranes from the endoplasmic reticulum (ER), in the form of pleiomorphic ER-to-Golgi carriers (EGCs). An important step in the biogenesis of the Golgi ribbon is the complete incorporation of the EGCs into the stacks. This requires the Golgi-matrix protein GM130, which continuously cycles between the cis-Golgi compartments and the EGCs. On acquiring GM130, the EGCs undergo homotypic tethering and fusion, maturing into larger and more homogeneous membrane units that appear primed for incorporation into the Golgi stacks. In the absence of GM130, this process is impaired and the EGCs remain as distinct entities. This induces the accumulation of tubulovesicular membranes, the shortening of the cisternae, and the breakdown of the Golgi ribbon. Under these conditions, however, secretory cargo can still be delivered to the Golgi complex, although this occurs less efficiently, and apparently through transient and/or limited continuities between the EGCs and the Golgi cisternae.     

3D organization and function of the cell: Golgi budding and vesicle biogenesis to docking at the porosome complex

Insights into the three-dimensional (3D) organization and function of intracellular structures at nanometer resolution, holds the key to our understanding of the molecular underpinnings of cellular structure-function. Besides this fundamental understanding of the cell at the molecular level, such insights hold great promise in identifying the disease processes by their altered molecular profiles, and help determine precise therapeutic treatments. To achieve this objective, previous studies have employed electron microscopy (EM) tomography with reasonable success. However, a major hurdle in the use of EM tomography is the tedious procedures involved in fixing, high-pressure freezing, staining, serial sectioning, imaging, and finally compiling the EM images to obtain a 3D profile of sub-cellular structures. In contrast, the resolution limit of EM tomography is several nanometers, as compared to just a single or even sub-nanometer using the atomic force microscope (AFM). Although AFM has been hugely successful in 3D imaging studies at nanometer resolution and in real time involving isolated live cellular and isolated organelles, it has had limited success in similar studies involving 3D imaging at nm resolution of intracellular structure-function in situ. In the current study, using both AFM and EM on aldehyde-fixed and semi-dry mouse pancreatic acinar cells, new insights on a number of intracellular structure-function relationships and interactions were achieved. Golgi complexes, some exhibiting vesicles in the process of budding were observed, and small vesicles were caught in the act of fusing with larger vesicles, possibly representing either secretory vesicle biogenesis or vesicle refilling following discharge, or both. These results demonstrate the power and scope of the combined engagement of EM and AFM imaging of fixed semi-dry cells, capable of providing a wealth of new information on cellular structure-function and interactions.     

Membrane biogenesis

The literary data on the problem of the membrane biogenesis are generalized. The mechanisms of formation, possible ways of metabolism of biomembrane structure in cells and the ways of their degradation are considered. A conclusion has been made on the existence in the cells several types of movement as for the separate components and membrane fragments as well.     

Peroxisome biogenesis

Peroxisome biogenesis conceptually consists of the (a) formation of the peroxisomal membrane, (b) import of proteins into the peroxisomal matrix and (c) proliferation of the organelles. Combined genetic and biochemical approaches led to the identification of 25 PEX genes-encoding proteins required for the biogenesis of peroxisomes, so-called peroxins. Peroxisomal matrix and membrane proteins are synthesized on free ribosomes in the cytosol and posttranslationally imported into the organelle in an unknown fashion. The protein import into the peroxisomal matrix and the targeting and insertion of peroxisomal membrane proteins is performed by distinct machineries. At least three peroxins have been shown to be involved in the topogenesis of peroxisomal membrane proteins. Elaborate peroxin complexes form the machinery which in a concerted action of the components transports folded, even oligomeric matrix proteins across the peroxisomal membrane. The past decade has significantly improved our knowledge of the involvement of certain peroxins in the distinct steps of the import process, like cargo recognition, docking of cargo-receptor complexes to the peroxisomal membrane, translocation, and receptor recycling. This review summarizes our knowledge of the functional role the known peroxins play in the biogenesis and maintenance of peroxisomes. Ideas on the involvement of preperoxisomal structures in the biogenesis of the peroxisomal membrane are highlighted and special attention is paid to the concept of cargo protein aggregation as a presupposition for peroxisomal matrix protein import. Electronic Publication     

p37 is a p97 adaptor required for Golgi and ER biogenesis in interphase and at the end of mitosis

We previously reported that p97/p47-assisted membrane fusion is important for the reassembly of organelles at the end of mitosis, but not for their maintenance during interphase. We have now identified a p97 adaptor protein, p37, which forms a complex with p97 in the cytosol and localizes to the Golgi and ER. siRNA experiments revealed that p37 is required for Golgi and ER biogenesis. Injection of anti-p37 antibodies into cells at different cell cycle stages showed that p37 plays an important role in both Golgi and ER maintenance during interphase as well as in their reassembly at the end of mitosis. In an in vitro Golgi reassembly assay, the p97/p37 complex has membrane fusion activity. In contrast to the p97/p47 pathway, this pathway requires p115-GM130 tethering and SNARE GS15, but not syntaxin5. Interestingly, although VCIP135 is also required, its deubiquitinating activity is unnecessary for p97/p37-mediated activities.