Global approaches to study Golgi function (original) (raw)
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Proteomics Characterization of Abundant Golgi Membrane Proteins
Journal of Biological Chemistry, 2001
A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and ␣ 2 P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.
Identification of new Golgi complex specific proteins by direct organelle proteomic analysis
PROTEOMICS, 2006
The Golgi complex is in the crossroad of the endocytic and secretory pathways. Its function is to post-translationally modify and sort proteins and lipids, and regulate the membrane balance in the cell. To understand the structure-function relationship of the Golgi complex the Golgi proteome has to be identified first. We have used a direct organelle proteomic analysis to identify new Golgi complex proteins. Enriched stacked Golgi membrane fractions from rat livers were isolated, and the proteins from these membranes were subsequently digested into peptides. The peptides were fractionated by cation-exchange chromatography followed by protein identification by automated capillary-LC/ESI-MS/MS analysis and database searches. Two different search programs, ProID and MASCOT were used. This resulted in a total of 1125 protein identifications in two experiments. In addition to the known Golgi resident proteins, a significant number of unknown proteins were identified. Some of these were further characterized in silico using different programs to provide insight into their structure, intracellular localization and biological functions. The Golgi localization of two of these newly identified proteins was also confirmed by indirect immunofluorescence.
The Journal of Cell Biology, 1991
Saccharomyces cerevisiae sec7 mutants exhibit pleiotropic deficiencies in the transit of proteins through the Golgi apparatus, and elaborate an array of Golgi apparatus-like cisternae at a restrictive growth temperature (37 degrees C). The SEC7 gene encodes an essential high-molecular weight protein (227 kD) that is phosphorylated in vivo. In cell lysates, Sec7 protein (Sec7p) is recovered in both sedimentable and soluble fractions. A punctate immunofluorescent pattern of Sec7p-associated structures seen in SEC cells coalesces in sec14 mutant yeast that accumulate exaggerated Golgi cisternae at 37 degrees C. Sec7p may function as a peripheral membrane protein that cycles between a soluble, cytosolic pool and a sedimentable, membrane-associated complex for its essential role in vesicular traffic through the Golgi apparatus. The transmembrane Kex2 protease, which processes precursors of secreted peptides within the yeast secretory pathway, is also localized by indirect immunofluoresce...
Single-population transcriptomics as a method to identify a network regulating Golgi structure
2021
a COPII vesicle is initiated by the activation of the small GTP-binding protein Sar1[8] by the guanine nucleotide-exchange factor Sec12. Sar 1 interacts directly to recruit Sec23 and Sec24. These proteins drive cargo capture by direct binding of cargo to Sec24 adaptor subunits and assemble the inner coat of the vesicle by forming tight heterodimers [9]. Many cargo proteins have specific signal sequences that mark them for COPII transport, while others may be incorporated via bulk flow [10]. The final step in building a COPII 1.1.2 Golgi to ER transport From the ERGIC compartment, ER resident proteins and membranes are recycled back to the ER. This is enabled by another set of vesicular coat proteins known as the COPI coat [15], which assembles on ERGIC and early Golgi membranes. The COPI coat is a heptameric structure comprising of COPA, COPB1, COPB2, COPD, COPE, COPG and COPZ subunits (α,β, β , γ, δ, , ζ). Subunits α,β , comprise the outer COPI coat, and the inner coat is formed by the β, δ, γ and ζ subunits [16]. This cytosolic protein complex is recruited by the GTP binding protein Arf-1 [17] which mediates the association of the coat proteins with the ERGIC/Golgi membranes in its GTP bound form. Arf-1 in turn requires activation by the Arf-Guanosine Exchange Factor (GEF). In Resident ER proteins usually carry a signal sequence that allow their retrieval into the ER. ER luminal proteins have a C-terminal KDEL sequence that is recognized by a KDEL receptor, which binds the COPI machinery to retrieve luminal ER proteins [18]. This receptor itself cycles between the ER and the Golgi Complex. Transmembrane proteins belonging to the ER are identified for retrieval using another signal sequence, characterized by the motif KKXX or KXKXX At the Trans-Golgi Network (TGN), mature proteins and lipids are sorted for their forward journey towards the Plasma Membrane, endosomes and secretory granules or for retrograde transport to earlier Golgi cisternae or the ER [27]. The TGN also receives cargo from various endosomes for retrograde transport, providing a point of convergence of the secretory and endocytic pathways [27]. More than five pathways have been described for transport of cargo from the TGN in the anterograde direction, and there are network interacting with the ERGIC compartments whereas the trans cisternae interact with the TGN [34]. Cisternae along the Golgi stack differ not only in location, but in their membrane compositions, thickness, pH, as well as the enzymes they house. This results in several gradients operating through a Golgi stack [35]. Individual Golgi stacks are encapsulated in a ribosome-free protein matrix called the 'compact zone' of the Golgi, the ribbon structure is closely dependent on the cytoskeleton and disruptions of actin or microtubule networks cause the ribbon to dismantle [41]. At either polar end (cis and trans faces), the Golgi associates with tubular networks such as Vesicular Tubular Structures (VTC's) on the cis side and the Trans-Golgi Network (TGN) on the trans side that link different organelles of the secretory pathway [42]. The Golgi Complex in plants and 1.2.2 The Golgi Complex as a dynamic organelle Despite the sophisticated architecture of the Golgi Complex, it is a highly dynamic organelle capable of rapid disassembly and reassembly. The most obvious case of this reorganization is during mitosis [50]. At the onset of mitosis, the Golgi ribbon is un-linked and peripheral membrane proteins are released into the cytoplasm. The individual Golgi stacks undergo unstacking and vesiculation [51]. This process is mediated by Arf1, and requires the phosphorylation of GM130 and GRASP65 by the mitotic kinases Cdk1 (Cyclindependent kinase 1) and Plk1 (Polo-like Kinase 1)[52][53]. Phosphorylation disrupts the tethering function of these proteins, causing Golgi disassembly [54][55]. Resulting Golgi vesicles are dispersed in the cytoplasm, and many are found to associate with astral microtubules at spindle poles. The Golgi membranes reform their original stacked organization after during telophase and cytokinesis, mediated by SNARE-led membrane fusion (dis-1.3.4 Signaling Platform The Golgi Complex acts as a signaling platform for a variety of cellular processes. These processes can either by related to Golgi structure and function, or be completely independent of trafficking. The Golgi responds to relayed signals originating outside the cell, cascades coming from other organelles as well as self-generated cues [76]. Plasma Membrane initiated signaling The typical Ras/MAPK (Mitogen Activated Protein Kinase) is triggered by growth factors binding to Plasma Membrane receptors. Growth factor stimulation can also lead to Ca2+ 1.4.2 Golgins Golgins are a family of proteins characterized by their extensively coiled-coil domains that are known to form a rod-like structure. They were originally identified as Golgi-localized auto-antigens cytoplasmic face of the Golgi [107]. Another feature that Golgins have in common is that they interact with small GTPases [36]. Golgins make up a large family of proteins that vary considerably in structure and function. The coiled-coil nature of Golgins make them ideal tethering proteins [108]. They are capable of linking membranes over relatively long distances, which allows for efficient capture of cargo between compartments. Tethering not only serves to capture cargo for traffic, but is also required for cisternae formation and ribbon linking [109]. Upon long-range capture of membranes by Golgins, a GTPase dependent conformational change in the coiled-coil region that enables the Golgin to bend in order to bring the target membrane in close contact with the recipient membrane. This would be followed by SNARE pairing and subsequent membrane fusion. Some Golgins can interact directly with SNARE proteins, others interact with other tethering complexes such as the COG and TRAPP complexes[110] [111]. The fusion of mainly COPI vesicles [123]. siRNA mediated depletion of Giantin was shown to cause increased cargo transport in conjunction with impaired glycosylation. Furthermore, depletion of Giantin also led to increased dispersion of Golgi stacks in nocadazole treated cells [124]. Although the exact role of this Golgin is yet to be elucidated, it is likely to play a role in both structural organization and glycosylation by the Golgi. 1.4.3 Trafficking Proteins Apart from the matrix proteins, there are many trafficking proteins and tethering complexes which regulate Golgi organization by mediating cargo flux in and out of the Golgi. Therefore, it is no surprise that the absence of these proteins has an effect on Golgi morphology. The main functions of a few of these complexes are highlighted below. Rab GTPases Rab GTPases consist of a family of 60 small Ras-like GTP-binding proteins that recruit a variety of trafficking proteins, owing to their ability to switch between GTP and GDP bound states [130]. About 20 Rabs are localized at the Golgi Complex, including Rab
The Golgi apparatus: an organelle with multiple complex functions
Biochemical Journal, 2011
Remarkable advances have been made during the last few decades in defining the organizational principles of the secretory pathway. The Golgi complex in particular has attracted special attention due to its central position in the pathway, as well as for its fascinating and complex structure. Analytical studies of this organelle have produced significant advances in our understanding of its function, although some aspects still seem to elude our comprehension. In more recent years a level of complexity surrounding this organelle has emerged with the discovery that the Golgi complex is involved in cellular processes other than the ‘classical’ trafficking and biosynthetic pathways. The resulting picture is that the Golgi complex can be considered as a cellular headquarters where cargo sorting/processing, basic metabolism, signalling and cell-fate decisional processes converge.
The Journal of Cell Biology, 2002
Multiprotein complexes are key determinants of Golgi apparatus structure and its capacity for intracellular transport and glycoprotein modification. Three complexes that have previously been partially characterized include (a) the Golgi transport complex (GTC), identified in an in vitro membrane transport assay, (b) the ldlCp complex, identified in analyses of CHO cell mutants with defects in Golgi-associated glycosylation reactions, and (c) the mammalian Sec34 complex, identified by homology to yeast Sec34p, implicated in vesicular transport. We show that these three complexes are identical and rename them the conserved oligomeric Golgi (COG) complex. The COG complex comprises four previously characterized proteins (Cog1/ldlBp, Cog2/ldlCp, Cog3/Sec34, and Cog5/GTC-90), three homologues of yeast Sec34/35 complex subunits (Cog4, -6, and -8), and a previously unidentified Golgi-associated protein (Cog7). EM of ldlB and ldlC mutants established that COG is required for normal Golgi mor...
Molecular basis for Golgi maintenance and biogenesis
Current Opinion in Cell Biology, 2004
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.
Golgi-IP, a novel tool for multimodal analysis of Golgi molecular content
The Golgi is a membrane-bound organelle that is essential for protein and lipid biosynthesis. It represents a central trafficking hub that sorts proteins and lipids to various destinations or for secretion from the cell. The Golgi has emerged as a docking platform for cellular signalling pathways including LRRK2 kinase whose deregulation leads to Parkinson disease. Golgi dysfunction is associated with a broad spectrum of diseases including cancer, neurodegeneration, and cardiovascular diseases. To allow the study of the Golgi at high resolution, we report a rapid immunoprecipitation technique (Golgi-IP) to isolate intact Golgi mini-stacks for subsequent analysis of their content. By fusing the Golgi resident protein TMEM115 to three tandem HA epitopes (GolgiTAG), we purified the Golgi using Golgi-IP with minimal contamination from other compartments. We then established an analysis pipeline using liquid chromatography coupled with mass spectrometry to characterize the human Golgi pr...
Cell biology of the endoplasmic reticulum and the Golgi apparatus through proteomics
Cold Spring Harbor perspectives in biology, 2013
Enriched endoplasmic reticulum (ER) and Golgi membranes subjected to mass spectrometry have uncovered over a thousand different proteins assigned to the ER and Golgi apparatus of rat liver. This, in turn, led to the uncovering of several hundred proteins of poorly understood function and, through hierarchical clustering, showed that proteins distributed in patterns suggestive of microdomains in cognate organelles. This has led to new insights with respect to their intracellular localization and function. Another outcome has been the critical testing of the cisternal maturation hypothesis showing overwhelming support for a predominant role of COPI vesicles in the transport of resident proteins of the ER and Golgi apparatus (as opposed to biosynthetic cargo). Here we will discuss new insights gained and also highlight new avenues undertaken to further explore the cell biology of the ER and the Golgi apparatus through tandem mass spectrometry.