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
    • "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.
    Journal of Cell Science 10/2015; DOI:10.1242/jcs.172395 · 5.43 Impact Factor
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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.
    Journal of Cell Science 09/2014; 127(22). DOI:10.1242/jcs.155408 · 5.43 Impact Factor
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    • "The cis-Golgi is a receiver of anterograde vesicles traveling from the ER, whereas the trans-Golgi is a departure site for vesicles traveling to endocytic compartments [1]. The ER resident proteins that escape to the Golgi and Golgi proteins cycle back to the ER via the retrograde pathway [2] [3] [4] [5] [6] [7] [8] [9]. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) [10] play a fundamental role in membrane fusion and have a polarized, gradient-like distribution in the Golgi. "
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    ABSTRACT: Two distinct sets of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) catalyze membrane fusion in the cis-Golgi and trans-Golgi. The mechanism that controls Golgi localization of SNAREs remains largely unknown. Here we tested three potential mechanisms, including vesicle recycling between the Golgi and the endoplasmic reticulum, partitioning in Golgi lipid microdomains, and selective intra-Golgi retention. Recycling rates showed a linear relationship with intra-Golgi mobility of SNAREs. The cis-Golgi SNAREs had higher mobility than intra-Golgi SNAREs, whereas vesicle SNAREs had higher mobility than target membrane SNAREs. The differences in SNARE mobility were not due to preferential partitioning into detergent-resistant membrane microdomains. We propose that intra-Golgi retention precludes entropy-driven redistribution of SNAREs to the endoplasmic reticulum and endocytic compartments.
    FEBS letters 06/2013; 587(15). DOI:10.1016/j.febslet.2013.06.004 · 3.17 Impact Factor
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