Recombinant Antibodies Against Subcellular Fractions Used to Track Endogenous Golgi Protein Dynamics in Vivo (original) (raw)
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Journal of Cell Biology, 2013
After leaving the endoplasmic reticulum, secretory proteins traverse several membranous transport compartments before reaching their destinations. How they move through the Golgi complex, a major secretory station composed of stacks of membranous cisternae, is a central yet unsettled issue in membrane biology. Two classes of mechanisms have been proposed. One is based on cargo-laden carriers hopping across stable cisternae and the other on “maturing” cisternae that carry cargo forward while progressing through the stack. A key difference between the two concerns the behavior of Golgi-resident proteins. Under stable cisternae models, Golgi residents remain in the same cisterna, whereas, according to cisternal maturation, Golgi residents recycle from distal to proximal cisternae via retrograde carriers in synchrony with cisternal progression. Here, we have engineered Golgi-resident constructs that can be polymerized at will to prevent their recycling via Golgi carriers. Maturation mod...
1998
We have addressed the question of whether or not Golgi fragmentation, as exemplified by that occurring during drug-induced microtubule depolymerization, is accompanied by the separation of Golgi subcompartments one from another. Scattering kinetics of Golgi subcompartments during microtubule disassembly and reassembly following reversible nocodazole exposure was inferred from multimarker analysis of protein distribution. Stably expressed ␣-2,6-sialyltransferase and N-acetylglucosaminyltransferase-I (NAGT-I), both C-terminally tagged with the myc epitope, provided markers for the trans-Golgi/trans-Golgi network (TGN) and medial-Golgi, respectively, in Vero cells. Using immunogold labeling, the chimeric proteins were polarized within the Golgi stack. Total cellular distributions of recombinant proteins were assessed by immunofluorescence (anti-myc monoclonal antibody) with respect to the endogenous protein, -1,4-galactosyltransferase (GalT, trans-Golgi/ TGN, polyclonal antibody). ERGIC-53 served as a marker for the intermediate compartment). In HeLa cells, distribution of endogenous GalT was compared with transfected rat ␣-mannosidase II (medial-Golgi, polyclonal antibody). After a 1-h nocodazole treatment, Vero ␣-2,6-sialyltransferase and GalT were found in scattered cytoplasmic patches that increased in number over time. Initially these structures were often negative for NAGT-I, but over a two-to threefold slower time course, NAGT-I colocalized with ␣-2,6-sialyltransferase and GalT. Scattered Golgi elements were located in proximity to ERGIC-53-positive structures. Similar trans-first scattering kinetics was seen with the HeLa GalT/␣-mannosidase II pairing. Following nocodazole removal, all cisternal markers accumulated at the same rate in a juxtanuclear Golgi. Accumulation of cisternal proteins in scattered Golgi elements was not blocked by microinjected GTP␥S at a concentration sufficient to inhibit secretory processes. Redistribution of Golgi proteins from endoplasmic reticulum to scattered structures following brefeldin A removal in the presence of nocodazole was not blocked by GTP␥S. We conclude that Golgi subcompartments can separate one from the other. We discuss how direct trafficking of Golgi proteins from the TGN/trans-Golgi to endoplasmic reticulum may explain the observed trans-first scattering of Golgi transferases in response to microtubule depolymerization.
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
A plasmid-based expression system to study protein-protein interactions at the Golgi in vivo
Analytical biochemistry, 2016
There is still an unmet need for simple methods to verify, visualize, and confirm protein-protein interactions in vivo. Here we describe a plasmid-based system to study such interactions. The system is based on the transmembrane domain (TMD) of the EF-hand Ca(2+) sensor protein calneuron-2. We show that fusion of 28 amino acids that include the TMD of calneuron-2 to proteins of interest results in prominent localization on the cytoplasmic side of the Golgi. The recruitment of binding partners to the protein of interest fused to this sequence can then be easily visualized by fluorescent tags.
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...
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.
GMAP-210, A Cis-Golgi Network-associated Protein, Is a Minus End Microtubule-binding Protein
Journal of Cell Biology, 1999
We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. M. Bornens. 1994. J. Cell Biol. 125:997-1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1,979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH 2 terminus binds to Golgi membranes. Overexpression of GMAP-210-encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH 2 -terminal domain associated with the cis-Golgi network whereas the COOHterminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus.