Tethering of transport vesicles in secretory and endocytic pathways: mechanisms and players View all 18 Articles. The Golgi apparatus is a central intracellular membrane organelle for trafficking and modification of proteins and lipids.
Its basic structure is a stack of tightly aligned flat cisternae. In mammalian cells, dozens of stacks are concentrated in the pericentriolar region and laterally connected to form a ribbon.
Despite extensive research in the last decades, how this unique structure is formed and why its formation is important for proper Golgi functioning remain largely unknown. Depletion of GRASPs in cells disrupts the Golgi structure and results in accelerated protein trafficking and defective glycosylation. In this minireview we summarize our current knowledge on how GRASPs function in Golgi structure formation and discuss why Golgi structure formation is important for its function.
The Golgi apparatus is a membrane-bound organelle found in all eukaryotic cells, including those of animals, plants, and fungi, and functions as a central hub in the exocytic secretory pathway Klute et al. The Golgi is the receiver of the entire output of the endoplasmic reticulum ER , where proteins and lipids are processed, sorted, and packaged into vesicles and transport carriers for delivery to their final destinations inside or outside of the cell.
Under electron microscope EM , the Golgi displays as stacks of flattened cisternae, which are often laterally linked into a ribbon-like structure in mammalian cells. By light microscopy, the Golgi is characterized by a compact reticular appearance located adjacent to the nucleus.
Despite the complexity, the Golgi structure is highly dynamic, and undergoes rapidly disassembly and reassembly during mitosis and under stress and physiological conditions Wang and Seemann, At the onset of mitosis, the Golgi disassembles into vesicles and tubular structures that are partitioned into the daughter cells, where they are reassembled into a new Golgi at the end of mitosis Shorter and Warren, The unique stacked morphology and dynamics of the Golgi have prompted numerous studies targeting the mechanisms of Golgi structure formation and function.
Morphological and biochemical research observed inter-cisternal proteinaceous connections that cross-link adjacent cisternae Figure 1A Turner and Whaley, ; Franke et al. Mild proteolysis removing these connections resulted in unstacking Cluett and Brown, Figure 1. A Electron micrograph of a Golgi apparatus from the green alga Chlamydomonas reinhardtii.
Cells were snap-frozen without chemical fixation, fractured and deep-etched Heuser, Arrows point to bridges between the cisternae. Scale bar, nm. GRASP55 has a similar domain structure. It is a peripheral protein on the cytoplasmic surface of the Golgi, directly targeted to the Golgi after synthesis in the cytosol Yoshimura et al. In a cell-free system that mimics Golgi disassembly and reassembly during the cell cycle, inhibition of GRASP65 using recombinant proteins or antibodies blocked the formation of Golgi stacks but not the generation of single cisternae Barr et al.
GRASPs are evolutionally conserved. GRASP orthologues and homologs have been identified in different species, including flies Kondylis et al. Most of these homologs are associated with the Golgi; however, some are also detected on other membranes. A number of labs have tested these proteins; some support their roles in Golgi stacking, while others provided alternative functions including Golgi ribbon linking, transport of specific cargo across the Golgi stack, unconventional secretion, cell cycle regulation, apoptosis, and cell migration, which have been summarized in a number of reviews Wang, ; Ramirez and Lowe, ; Wei and Seemann, ; Vinke et al.
Most recently, new findings have been made on GRASPs, including available crystal structures, identification of novel GRASP interacting proteins, and new insights between Golgi structure formation and function, which have triggered us to update our understanding of GRASPs in Golgi structure formation and function. This dual anchoring of GRASPs onto membrane restricts the orientation of the protein to favor trans pairing over cis Bachert and Linstedt, , thus ensuring membrane tethering by forming trans -oligomers Wang et al.
Second, GRASPs oligomerization is regulated by phosphorylation, which provides an explanation for Golgi disassembly and reassembly during cell division Tang and Wang, In cells, inhibition of mitotic kinases blocked mitotic Golgi fragmentation Misteli and Warren, ; while microinjection of mitotic kinases such as Cdk1 and polo-like kinase Plk led to Golgi disassembly Wang et al.
In vitro , treatment of purified Golgi stacks with mitotic kinases resulted in cisternal unstacking Wang et al. These results demonstrate that Golgi structure formation is regulated by phosphorylation during the cell cycle.
There are some differences between these reports on the arrangements of the GRASP domain, possibly because of the differences in the protein length used in the studies and the addition of a GM peptide that may cause conformational change. None of the structural studies were able to include the SPR domain, and thus the structural basis of phosphorylation regulation of GRASP oligomerization remains unknown.
These results suggest GRASPs as ideal candidates in Golgi stacking than the long coiled-coil golgins, which are better known for membrane tethering Wong and Munro, When GRASP65 is coated onto the surface of beads, it causes the beads to aggregate, demonstrating that it can directly link surfaces together Wang et al. Simultaneous depletion of both caused complete disassembly of the Golgi stacks Xiang and Wang, Conversely, expression of non-phosphorylatable GRASP65 mutants enhanced Golgi stacking in interphase and inhibited Golgi fragmentation in mitosis Tang et al.
While this hypothesis indicates a high complexity in Golgi stacking, it helps explain how Golgi stacking occurs in organisms such as plant in which no GRASP proteins have been identified. While the different observations may be due to distinct experimental systems, the knockdown efficiency, and the approaches used to analyze the effects of GRASP deletion.
In fact, these two observations are not mutually exclusive, and it is possible that GRASPs function in both Golgi stacking and ribbon linking by forming trans -oligomers.
Given that the gaps between Golgi stacks are much larger and more heterogeneous 10s—s nm than the distance between cisternae within each stack Cluett and Brown, , it is possible that other bridging proteins may help GRASPs in ribbon linking, of which golgins are ideal candidates because of their long coiled-coil domains known in membrane tethering.
Consistent with this idea, inhibition by RNAi-mediated depletion or microinjection of antibodies of GM Puthenveedu et al. Depletion of Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro , Mena, and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion.
To a great extent, organelle function relies on its structure. However, why Golgi stack formation is important for its function has been remaining largely as a mystery in the field for many decades. Golgi cisternae do not normally form stacks in budding yeast Saccharomyces cerevisiae , suggesting that stacking is not absolutely required for cell survival. However, Golgi stacking is a pronounced feature in all metazoans and many unicellular eukaryotes, implying that it has important functional consequences.
First, stacking may impact protein trafficking. The close spatial arrangement of cisternae in stacks minimizes the distance that molecules must travel; local tethering proteins facilitate vesicle fusion with Golgi membranes Lupashin and Sztul, , therefore stacking should enhance protein trafficking. However, stacking restricts the surface for vesicle budding and fusion to the rims of the cisternae and so it may retard trafficking. Thus, this relationship is still not well understood.
Second, stacking may be required for accurate glycosylation. The Golgi harbors various glycosyltransferases and glycosidases in different sub-compartments. An ordered structure is likely required to carry out precise, sequential modifications as cargo proteins pass between cisternae Kornfeld and Kornfeld, ; Varki, ; Roth, In yeast and other fungi, N-glycosylation in the Golgi mainly involves the addition of mannoses Wildt and Gerngross, In multi-cellular organisms, N-glycosylation of membrane and secretory proteins is more complex and critical.
Accurate glycosylation is essential for their cellular functions, including cell adhesion and migration, cell-cell communication, and immunity Ohtsubo and Marth, In polarized cells such as neurons and epithelial cells, N- and O-linked glycosylations serve as apical sorting signals Weisz and Rodriguez-Boulan, This may explain why stacking is not required in yeast, but is essential for life in higher order organisms.
Third, stacking may ensure that sorting occurs only when cargo molecules reach the trans -Golgi network TGN but not in earlier sub-compartments. The best way to answer these questions is to disrupt the Golgi stacks and assess the subsequent effects. One surprising observation is that Golgi destruction accelerates protein trafficking. How could Golgi unstacking enhance protein trafficking? A plausible explanation is that unstacking increases the accessibility of coat proteins to Golgi membranes for vesicle budding and fusion, thereby increasing the rate of protein transport Figure 2.
Figure 2. Golgi destruction accelerates protein trafficking and impairs accurate glycosylation and sorting. When Golgi cisternae are fully stacked A , vesicles can only form and fuse at the rims. This slows down trafficking, but enforces accurate glycosylation. Once the cisternae are unstacked B , more membrane surface area becomes accessible for vesicle budding and fusion, thereby increasing cargo transport.
This, however, causes glycosylation and sorting defects adapted and modified from Xiang et al. Golgi destruction impairs accurate protein glycosylation and sorting.
GRASP depletion resulted in decreased sialic acid on the cell surface, but the expression level and localization of Golgi enzymes did not significantly change Xiang et al. This effect was confirmed by analysis of individual glycoproteins, flow cytometry of cells surface-stained by fluorescent lectins, and glycomic studies.
These results indicate that Golgi structure formation is required for accurate protein glycosylation and sorting. One reasonable explanation is that stacking controls the sequence and speed of protein transport through the Golgi, allowing the cargo to remain in each sub-compartment for a sufficient time period to ensure proper glycosylation in the stack and proper sorting at the TGN; unstacking increases the membrane surface for vesicle formation and protein transport, but causes glycosylation and sorting defects Figure 2.
When one GRASP protein was substituted by the other, the Golgi ribbon was intact, but the membranes were decompartmentalized and glycosylation became defective. Thus, each GRASP plays a cisterna-specific role in linking ministacks to ensure Golgi compartmentalization, enzymes localization, and proper glycosylation Jarvela and Linstedt, Additionally, cells from the GRASP65 knockout mouse also showed defects in cis -Golgi integrity and glycosylation in the plasma membrane Veenendaal et al.
Significant progress has been made in the last few years on the GRASP proteins, including their biochemical properties, phosphorylation regulation, crystal structures, and interacting partners, which support GRASPs as the best candidates for Golgi stacking factors. However, there have been discrepancies on their roles in Golgi structure formation mostly resulting from RNAi depletion experiments. Rabouille and colleagues used traditional mouse gene knockout technology to delete GRASP65, finding that such mice are viable with no apparent physiological deficits or gross morphological perturbations of the Golgi Veenendaal et al.
In their recent study Grond et al. Next, using a conditional knockout approach, double GRASP null cells were produced postnatally in the small intestine, and the Golgi of intestinal epithelial cells was examined. In these cells, stacked Golgi cisternae were observed, but their arrangement into a ribbon was compromised, a result corroborated by more detailed analysis of cells in organoid cultures.
These findings are at odds with the conclusions of Wang and colleagues Bekier et al. One possible reason for the disparities between these two studies is that Bekier et al.
Analyses of siRNA-depleted and gene-edited cell lines and modified animals are often complicated by incomplete depletion of a query protein, unintended loss of other proteins, or compensatory processes that obscure loss-of-function effects. Notably, siRNA depletion of GM, which is associated with GRASP65 on the cis cisterna, impairs secretory traffic from the endoplasmic reticulum to the Golgi apparatus, resulting in a reduction in the size of Golgi cisternae and diminished interstack connectivity possibly due to vesiculation of cisternae Seemann et al.
Hence, the acute effects of GRASP protein depletion could be determined before the onset of potentially confounding effects. Fluorescence recovery after photobleaching assays of a fluorescently tagged Golgi resident protein revealed that acute depletion of both GRASP65 and GRASP55 resulted in decreased mobility of the resident Golgi enzyme within the ribbon, indicating that connectivity of cisternae between stacks was compromised.
Similar to Zhang and Seemann , they observed that the Golgi ribbon was disassembled upon inactivation of GRASP proteins, but stacking of cisternae was unaffected. Taken together, these results conclusively show that acute depletion of GRASP65 and GRASP55 impairs lateral linking of stacked Golgi cisternae within the ribbon while not affecting stacking of cisternae. These new studies suggest that the integrity of the Golgi matrix critically depends on the presence of GRASP proteins, and their absence perturbs the balance of cargo flow through the Golgi, reducing the interstack exchange required to maintain connectivity of stacks within the ribbon.
Hence, the GRASP proteins are positioned at the vesicle-rich interface between adjacent cisternal stacks. Grond et al. These new studies firmly shift our view of GRASP protein function away from the stacking of Golgi cisternae, and we look forward to new mechanistic insights into the roles of GRASP proteins in Golgi ribbon formation as well as in non—Golgi-dependent processes, such as unconventional protein secretion Kinseth et al.
The organization of the Golgi apparatus in vertebrate cells. Individual stacks of Golgi cisternae are aligned side to side to form the Golgi ribbon. The GRASP65 and GRASP55 proteins are depicted to be enriched on the rims of the indicated cisternae within individual stacks of cisternae, where they are required to maintain the arrangement of stacks into the ribbon.
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By: Pamela L. Connerly, Ph. Citation: Connerly, P. Nature Education 3 9 The Golgi apparatus transports and modifies proteins in eukaryotic cells. How have scientists studied dynamic protein movements through the Golgi? Aa Aa Aa. Figure 2: Two models of protein trafficking through the Golgi. A The cisternal maturation model of protein movement through the Golgi.
The Vesicular Transport Model: Evidence. The Cisternal Maturation Model. Which Model Is More Accurate? Figure 3: Cisternal maturation in Golgi of Saccharomyces cerevisiae. Golgi cisternae were labeled with dyes to track their movement over time in individual yeast cells. A recent gathering of prominent Golgi researchers identified several important questions to be addressed in the future, including: Do different types of secretory cargo follow distinct routes through the Golgi?
What molecular mechanisms drive and regulate cisternal maturation? Are there specialized domains in the Golgi cisternae?
How are they created, and what roles do they play in cargo sorting and export? How are the Golgi compartments constructed and remodeled? Is Golgi stacking fundamentally important for membrane traffic? If so, how do organisms such as S. References and Recommended Reading. Article History Close.
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