Scales, S. J., Pepperkok, R. & Kreis, T. E. Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI. Cell 90, 1137-1148

Department of Cell Biology, University of Geneva Sciences III, Switzerland.
Cell (Impact Factor: 32.24). 10/1997; 90(6):1137-48. DOI: 10.1016/S0092-8674(00)80379-7
Source: PubMed


Exocytic transport from the endoplasmic reticulum (ER) to the Golgi complex has been visualized in living cells using a chimera of the temperature-sensitive glycoprotein of vesicular stomatitis virus and green fluorescent protein (ts-G-GFP[ct]). Upon shifting to permissive temperature, ts-G-GFP(ct) concentrates into COPII-positive structures close to the ER, which then build up to form an intermediate compartment or transport complex, containing ERGIC-53 and the KDEL receptor, where COPII is replaced by COPI. These structures appear heterogenous and move in a microtubule-dependent manner toward the Golgi complex. Our results suggest a sequential mode of COPII and COPI action and indicate that the transport complexes are ER-to-Golgi transport intermediates from which COPI may be involved in recycling material to the ER.

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Available from: Suzie J Scales, Apr 27, 2015
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    • "It has been known for 30 years that microtubule depolymerization (for instance with nocodazole or colchicine) alters the Golgi morphology of mammalian cells. Instead of the single copy organelle capping the nucleus in non-treated cells, the Golgi ribbon appears fragmented leading to single stacks or group of stacks (Ellinger and Pavelka, 1984; Rogalski and Singer, 1984; Turner and Tartakoff, 1989; Cole et al., 1996; Scales et al., 1997). It is also increasingly recognized that in addition to the centrosome, the Golgi is a major site of microtubule formation in various cell types (Chabin-Brion et al., 2001; Efimov et al., 2007; Miller et al., 2009), including hippocampal neurons (Stiess et al., 2010; Yau et al., 2014) and motor neurons (Bellouze et al., 2014), see for review (Zhu and Kaverina, 2013; Rios, 2014; Kapitein and Hoogenraad, 2015; Sanders and Kaverina, 2015). "
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    ABSTRACT: Pathological alterations of the Golgi apparatus, such as its fragmentation represent an early pre-clinical feature of many neurodegenerative diseases and have been widely studied in the motor neuron disease amyotrophic lateral sclerosis (ALS). Yet, the underlying molecular mechanisms have remained cryptic. In principle, Golgi fragmentation may result from defects in three major classes of proteins: structural Golgi proteins, cytoskeletal proteins and molecular motors, as well as proteins mediating transport to and through the Golgi. Here, we present the different mechanisms that may underlie Golgi fragmentation in animal and cellular models of ALS linked to mutations in SOD1, TARDBP (TDP-43), VAPB, and C9Orf72 and we propose a novel one based on findings in progressive motor neuronopathy (pmn) mice. These mice are mutated in the TBCE gene encoding the cis-Golgi localized tubulin-binding cofactor E, one of five chaperones that assist in tubulin folding and microtubule polymerization. Loss of TBCE leads to alterations in Golgi microtubules, which in turn impedes on the maintenance of the Golgi architecture. This is due to down-regulation of COPI coat components, dispersion of Golgi tethers and strong accumulation of ER-Golgi SNAREs. These effects are partially rescued by the GTPase ARF1 through recruitment of TBCE to the Golgi. We hypothesize that defects in COPI vesicles, microtubules and their interaction may also underlie Golgi fragmentation in human ALS linked to other mutations, spinal muscular atrophy (SMA), and related motor neuron diseases. We also discuss the functional relevance of pathological Golgi alterations, in particular their potential causative, contributory, or compensatory role in the degeneration of motor neuron cell bodies, axons and synapses.
    Full-text · Article · Dec 2015 · Frontiers in Neuroscience
    • "The intensities of these structures increased gradually over time before the structures segregated from the ER and moved towards the Golgi complex (Fig. 3B; see also Refs. (Presley et al., 1997; Runz et al., 2006; Scales et al., 1997) and supplementary movies 1 and 2). The time between the first appearance of these structures and their release from the ERES varied considerably between a few seconds and several minutes (data not shown). "
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    ABSTRACT: Newly synthesized proteins are sorted into COPII-coated transport carriers at the endoplasmic reticulum (ER). Assembly of the COPII coat complex, which occurs at ER exit sites (ERES), is initiated by membrane association and GTP loading of SAR1, followed by the recruitment of the SEC23/24 and SEC13/31 sub-complexes. Both of these two sub-complexes stimulate GTP hydrolysis and coat disassembly. This inherent disassembly capacity of COPII complexes needs to be regulated to allow sufficient time for cargo sorting and transport carrier formation. Using fluorescence recovery after photobleaching (FRAP) and mathematical modelling we show that p150(glued), a component of the dynactin complex, stabilizes the COPII pre-budding complex on ER membranes in a microtubule-independent manner. Concentration of the secretory marker ts-O45-G at ERES is reduced in the presence of a C-terminal p150(glued) fragment that prevents binding of endogenous p150(glued) to SEC23. A similar cargo reduction is observed upon p150(glued) knockdown. Altogether, our data suggest that cargo concentration at ERES is regulated by p150(glued) to coordinate protein sorting and transport carrier formation with the subsequent long-range transport towards the Golgi complex along microtubules.
    No preview · Article · Oct 2015 · Journal of Cell Science
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    • "r results agree with the notion that EB1 complexes formed with other +TIPs mediate the attachment of microtubule tips to subcellular targets . Efficient ER - to - Golgi trafficking requires microtubules on which secretory cargos move towards the Golgi in association with the minus - end - directed motor dynein – dynactin ( Presley et al . , 1997 ; Scales et al . , 1997 ; Watson et al . , 2005 ) . Our results showed that MMG8 is required for microtubule - dependent ER - to - Golgi trafficking ( Fig . 2C , E ) . Interestingly , this function of MMG8 required its binding to EB1 or EB3 and thus the association of EB1 or EB3 with the Golgi ( Fig . 8 ) . We envision that the EB1 / EB3 - mediated attachment "
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    ABSTRACT: The Golgi apparatus of mammalian cells is known to be a major microtubule-organizing site that requires microtubules for its organization and protein trafficking. However, the mechanisms underlying the microtubule organization of the Golgi apparatus remain obscure. We used immunoprecipitation coupled with mass spectrometry to identify a widely expressed isoform of the poorly characterized muscle protein myomegalin. This novel isoform, myomegalin variant 8 (MMG8), localized predominantly to cis-Golgi networks by interacting with AKAP450, and this interaction with AKAP450 was required for the stability of both proteins. Disrupting MMG8 expression affected ER-to-Golgi trafficking and caused Golgi fragmentation. Furthermore, MMG8 associated with γ-tubulin complexes and with the microtubule plus-end tracking protein EB1, and MMG8 was required for the Golgi localization of these 2 molecules. On the Golgi, γ-tubulin complexes mediated microtubule nucleation, whereas EB1 functioned in ER-to-Golgi trafficking. These results indicate that MMG8 participates in Golgi microtubule organization and thereby plays a crucial role in the organization and function of the Golgi apparatus.
    Preview · Article · Sep 2014 · Journal of Cell Science
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