Seminars in Cell & Developmental Biology 18 (2007) 435–447 Review COPII under the microscope Semra J. Kirk, Theresa H. Ward ∗ Immunology Unit, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK Available online 12 July 2007 Abstract Transport through the secretory pathway begins with COPII regulation of ER export. Driven by the Sar1 GTPase cycle, cytosolic COPII proteins exchange on and off the membrane at specific sites on the ER to regulate cargo exit. Here recent developments in COPII research are discussed, particularly the use of live-cell imaging, which has revealed surprising insights into the coat’s role. The seemingly static ER exit sites are in fact highly dynamic, and the ability to visualise trafficking processes in intact living cells has highlighted the adaptable nature of COPII in cargo transport and the emerging roles of auxiliary factors. © 2007 Elsevier Ltd. All rights reserved. Keywords: COPII; Microscopy; GFP; Secretory pathway; ER exit Contents 1. 2. 3. 4. 5. ∗ Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Spatial organisation of ER exit sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Organisation of conserved COPII components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cargo travels through ER exit sites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. ER exit site association with microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . COPII and cargo in living cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. COPII coat dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Cargo release alters COPII kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. COPII kinetics and the lipid environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Cargo modulation of ERES organisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ER exit site biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Changes in ERES number during the cell cycle and self-organisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. ‘Early’ COPII components with putative role in tER organisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Sec12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Sec16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. Cooperation of Sec16 and Sar1 in mammalian cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.4. p125 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.5. p115 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. The role of COPII in Golgi biogenesis and maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Corresponding author. Tel.: +44 207 612 7869; fax: +44 207 927 2807. E-mail addresses: semra.kirk@lshtm.ac.uk (S.J. Kirk), theresa.ward@lshtm.ac.uk (T.H. Ward). 1084-9521/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2007.07.007 436 436 436 438 439 439 439 439 440 440 440 440 441 441 442 442 442 443 443 444 444 444 447 436 S.J. Kirk, T.H. Ward / Seminars in Cell & Developmental Biology 18 (2007) 435–447 1. Introduction The COPII coat complex is a key determinant in the fidelity of sorting and transport of newly synthesised biosynthetic cargo out of the endoplasmic reticulum (ER) and on towards the Golgi from where it can be directed to the plasma membrane. As described elsewhere in this issue, this coat comprises the small GTPase Sar1, and the heterodimers Sec23/Sec24 and Sec13/Sec31. Recruitment of the COPII subunits is sequential, as defined biochemically, with the activation of Sar1 to its GTP-bound form, by the membrane-bound nucleotide exchange factor Sec12, enabling downstream recruitment of the remaining components [1,2]. This review focuses on the aspects of COPII morphology and function that have been determined by cell biological methodology. erally [7,15]. Immuno-electron microscopy (EM) showed that COPI and COPII labelling at the ER–Golgi interface does not overlap—COPII is found at tER budding sites while COPI is found on the membrane buds at VTC membranes [7,16]. The continuity or vesicular nature of the membranes of the ER exit site has been the subject of some debate. The budding profiles visualised originally in thin sections [3,14] may have missed the tubular nature of the complexes [5,7,17,18] where COPII components are clearly visible on tubules emanating from the ER [17] (Fig. 1). However, it is apparent that some COPII-coated membranes are distinct from the ER as shown using immunoelectron tomography [19]. This may reflect a temporary scission from the ER prior to fusion with existing tubular structures in the VTC. 2.1. Organisation of conserved COPII components 2. Spatial organisation of ER exit sites In mammalian cells the zones of protein export from the endoplasmic reticulum (ER) are defined through microscopy as ribosome-free regions that label for the COPII coat complex [3,4]. The COPII-coated region or budding zone is termed the transitional ER (tER; [3,5–8]) while the adjacent vesicular–tubular clusters (VTCs), also known as the ER–Golgi intermediate compartment (ERGIC) or pre-Golgi intermediates [9,10], are labelled for the COPI coat [7,11], a second multisubunit coat complex that is required for sorting and transport both at ER exit sites and at the Golgi [12,13]. For the purpose of this review we define these membranes in their entirety, i.e. comprising COPII and COPI label, as the peripheral ER exit site (ERES) or ER export domain (Fig. 1, [9]), while some literature refers to the tER region alone as the ERES. Many tER directly appose the cis-Golgi stack [14] and this ER–Golgi interface shows some evidence of membrane interconnectivity [5]. In the region of 50–60% of ER export sites are actively positioned in the perinuclear region close to the microtubule organising centre (MTOC) while others are distributed periph- While the COPII machinery is shared between all eukaryotes, including yeast, plants and mammals, and appears to fulfil the same selective role in ER export, the morphological organisation varies greatly between organisms. In the yeast Saccharomyces cerevisiae the COPII components appear to be distributed evenly throughout the cytoplasm as shown by expression of a green fluorescent protein chimaera of Sec13 (Sec13-GFP), which localises to 30–50 spots per cell [20]. These spots may potentially represent individual COPII vesicles, as the fluorescence of multiple copies of the protein in a concentrated area would give rise to a detectable fluorescent signal despite a 50 nm COPII-coated vesicle being below the limit of resolution by light microscopy [20]. This pattern is replicable for the other COPII heterodimer subunits, suggesting that ER export in S. cerevisiae can occur from any point on the ER membrane [1,20]. In contrast, the diploid yeast Pichia pastoris and some other lower eukaryotes, many plant models, and all mammalian cells show localisation of COPII components to discrete subdomains of the ER [20,21]. How these organised tER sites then link to the Golgi, the organelle downstream in the secretory pathway, varies greatly. Fig. 1. Ultrastructure of ER exit sites. (A) Diagrammatic representation of a peripheral ER exit site. The COPII-coated budding regions (green) are also known as transitional ER or tER, while the COPI-coated tubular-vesicular membranes (red) are also known as the ERGIC or VTCs (after [9,17,19]). (B) Immuno-EM of a peripheral ER exit site. NRK cells stably expressing Sec13-YFP (a COPII component) were fixed and immunogold-labelled with anti-GFP antibodies. This figure shows defined ER membranes not labelled for Sec13 closely apposed to a cluster of membranes, which peripherally label for Sec13. Reproduced from [29] with permission from Dr. J. Lippincott-Schwartz. S.J. Kirk, T.H. Ward / Seminars in Cell & Developmental Biology 18 (2007) 435–447 For example, P. pastoris shows organisation of the tER into two to six large spots per cell [20] and organised tER sites are detectable by immuno-EM juxtaposed to ordered Golgi stacks. Therefore this organisation of the tER may translate into greater organisation of Golgi structure, as the Golgi in S. cerevisiae exists as individual mobile cisternae throughout the cytoplasm with early and late Golgi markers segregated [22], while P. pastoris Golgi are stacked at ER exit sites. Similarly the protozoan parasites Toxoplasma gondii and Trypanosoma brucei have a single stacked Golgi, which is closely associated with a single ER exit site [23,24]. Plant cells contain many individual Golgi stacks, which are each associated with a tER site, and these move together along the ER, presumably a mechanism for facilitating cargo sorting and export [21]. In mammalian cells several immunolocalisation studies have revealed that the ER exit sites are distributed as multiple discrete punctate structures throughout the cytosol while the stacked Golgi apparatus is found at the MTOC [6,8,25,26]. Colocalisation of Sec13 with an ER marker showed that these structures are always closely associated with ER tubules but are specifically distinct from the ER and therefore that Sec13 labels only tER sites, a subdomain of the ER [27] (Fig. 2). Only partial overlap with proteins of the ERGIC is found—by confocal microscopy a close apposition of markers for COPII and ERGIC-53, a protein that cycles through the early secretory pathway and colocalises with COPI in the VTCs [10,28], is found but with clearly distinct distributions [27,29]. 437 This is confirmed by comparative EM where immunolocalised Sec13 or Sec23 is only found in the immediate vicinity of ER tubules, while ERGIC-53 is found to label membranes throughout the ERES [28,29]. Similarly KDEL receptor, which also cycles between the ER and Golgi recycling proteins bearing the KDEL sequence to the ER [30], is found homogeneously distributed through the ER exit site and only a small proportion shows colocalisation with COPII [16]. This differential distribution between COPII proteins and markers of the ERGIC is not defined in other organisms where the COPII-labelled tER immediately abuts the Golgi. Therefore while COPII components are evolutionarily conserved, and their roles in cargo transport preserved, their cellular distribution varies considerably and how this affects their behaviour has been the subject of interest. The advent of GFP as a tool for cell biologists has enabled greater definition of the role and behaviour of COPII. In mammalian cells, similar punctate localisation patterns have been described for GFP chimaeras of all the COPII components [26,27,31,32]. This has enabled dissection of the in vivo dynamics of the tER and the coat proteins themselves. Time-lapse imaging of COPII-GFP chimaeras in mammalian cells demonstrated that COPII-labelled membranes are relatively immobile and long-lived [27,31]. Only rarely do COPII-labelled puncta exhibit a single rapid movement of more than 1 ␮m [27,31]. Movements in ERES appear to reflect movement of the underlying ER rather than motor-driven transport of the tER itself Fig. 2. Immunofluorescence of ER exit sites in COS cells labelled for the COPII subunit Sec31 (green) for comparison of localisation to either microtubules or ER (purple). Inset box shows magnified region demonstrating close alignment of ER exit sites to both. 438 S.J. Kirk, T.H. Ward / Seminars in Cell & Developmental Biology 18 (2007) 435–447 and this was demonstrated by co-imaging Sec24D-YFP with CFP-ER where Sec24D-labelled puncta co-align with ER at all times and slight movements of the COPII label coincide with movement of the associated reticular network [31]. In the case of the longer-range movements this is unlikely to be dependent on ER movement as they are abolished by treatment with nocodazole (which prevents microtubule polymerisation) indicating that these are microtubule dependent [31] but the function of these larger movements is unknown. In contrast to COPIIlabelled puncta, proteins of the ERGIC such as ERGIC-53 and COPI show long-range rapid movements that are microtubuledependent [29,33–35]. Therefore live-cell imaging gave rise to the important observation that COPII remains at static peripheral domains and does not associate with pre-Golgi intermediates trafficking in towards the Golgi. 2.2. Cargo travels through ER exit sites Localisation of distinct puncta of COPII fluorescence suggests a fixed site which supports multiple budding events (as previously seen in EM). Recruitment of cargo from the ER into a functional ER-to-Golgi carrier might be expected to result in expenditure of the ER export sites. The temperature sensitive variant (ts045) of vesicular stomatitis virus G protein (VSVG) has been widely used as a model cargo to study secretory transport in mammalian cells [36]. ts045-VSVG (hereafter VSVG) reversibly misfolds and is retained in the ER at 40 ◦ C, but upon shifting to the permissive temperature (32 ◦ C) the protein folds and is transported out of the ER, a property unaffected by addition of a GFP moiety [37,38]. An initial study which used rsec22b-GFP, a mammalian SNARE protein involved in regulation of ER-to-Golgi transport [39], showed first the accumulation of VSVG from the ER pool into punctate structures labelled for rsec22b, which then pulled off to the Golgi without associated rsec22b [40]. Although rsec22b shows greater mobility than COPII-labelled tER and is therefore not a true tER marker, this study demonstrated that the ERES was able to support cargo transport and was not consumed by the cargo trafficking. Experiments looking at the passage of the cargo protein VSVG-FP through Sec24D-FP labelled sites showed that upon release of cargo, VSVG puncta that initially colocalised with Sec24D pulled off and tracked in towards the Golgi leaving behind a clear COPII-labelled puncta [31], similarly seen with export of procollagen [41]. Importantly this general immobility suggests that the ERES are long-lived despite the passage of cargo [31,40]. Moreover, the same ERES can be reused by repeated cargo export cycles ([42], Ward, unpublished observations). Pepperkok and coworkers found that individual ERES released VSVG carriers one to four times during one wave of cargo export as defined by a sharp drop in fluorescence intensity at the same position [42]. The translocation of cargo into the Golgi requires the sequential coupling of COPII to the COPI coat. The nature of this handover is not clear but the requirement for COPII activity to precede COPI was demonstrated first in a permeabilised cell system [43] and then later by looking at fixed timepoints for VSVG colocalisation with either COPII- or COPI-labelled spots [38]. Quantitation of this localisation showed clearly that the majority of VSVG colocalised with COPII at the earlier timepoints but that this diminished as the amount of VSVG associated with COPI increased, and that it could be found in structures that colabelled for both coats [38]. Live imaging of Sec24D-GFP and COPI, visualised through microinjection of a fluorescently labelled COPI-specific antibody which does not interfere with COPI function, has shown that COPI spots initially localised with COPII then move off to the Golgi, leaving the COPII structure intact [31]. Furthermore there is no evidence that COPI intermediates traffic in to the Golgi with COPII label, showing that these coats act consecutively in anterograde transport between the ER and the Golgi. Low temperature blocks have been used to separate the early transport steps. At 10 ◦ C VSVG can fold and exit the ER into COPII-labelled puncta but does not enter VTCs labelled for COPI [44]. These early transport intermediates are viable for trafficking to the Golgi but, if subjected to nonpermissive VSVG folding temperatures, VSVG returns to the ER [44]. When VSVG is collected into the 15 ◦ C compartment (equivalent to COPI-bearing VTCs), return to the nonpermissive temperature results in VSVG continuing to the Golgi [44]. Therefore VTCs do not support VSVG unfolding while the tER sites are available for quality control and can return cargo directly to the reticular ER. Mammalian Sar1 (or SAR1A [45]) can modulate the incorporation of cargo into nascent transport carriers—different cargos display different requirements for GTP hydrolysis by Sar1. Model cargos used in this context include small membranebound proteins such as VSVG-GFP, or GPI-GFP which is targeted to the ER membrane through a lipid anchor (a glycosyl phosphatidylinositol (GPI) moiety) [46,47], and soluble cargo such as lum-GFP (GFP targeted to the ER lumen by addition of a cleavable signal sequence) [48] or large lumenal cargo such as procollagen [41]. VSVG and lum-GFP can readily accumulate into membranes induced by GTP-restricted Sar1 expression, whereas GPI-GFP is retained in the ER, and is only temporarily able to exit the ER [49]. While Sar1 activity is required for procollagen export from the ER [41,50], light and electron microscopy has revealed that PC-containing membrane domains are seen to form from the ER at points adjacent to COPII-coated ERES, suggesting that this large cargo is exported by en bloc protrusion from the ER [17,51]. PC and VSVG are proposed to export from the ER in different carriers that then converge in VTCs before trafficking on to the Golgi [17]. Whether procollagen, as a lumenal cargo, exits the ER by bulk flow, as proposed for chymotrypsinogen or amylase [16], or requires an additional export receptor has yet to be resolved [17]. A recent review discusses more fully how the transport of large cargo might be mediated [52]. How COPII might regulate the differential requirements of the cargo export through the tER is unclear but rather than the historical dogma of COPII involvement in biogenesis of transport vesicles, it may have a more complex role of organisation and regulation of export. This could be through the formation of specialised membrane domains for cargo export, analogous to the proposed role for membrane domain formation of COPI [34]. S.J. Kirk, T.H. Ward / Seminars in Cell & Developmental Biology 18 (2007) 435–447 2.3. ER exit site association with microtubules Positioning of intracellular organelles is dependent on organisation of the cytoskeleton, and microtubules in particular determine the directionality of traffic through the secretory pathway, although they are not essential to biosynthetic protein transport [53–55]. The radial assembly of microtubules in mammalian cells emanating from the MTOC is polarised such that Golgi stacks are clustered together in a juxtanuclear array at the minus ends of microtubules while tER sites are closely associated along the length of microtubules [32] (Fig. 2). There is a clear circumstantial link between COPII and microtubules—Sar1-GTP (the GTP-restricted mutant of Sar1) causes reorganisation of ER exit sites to the perinuclear region clustering around the MTOC [29,49,56]. The slow translocation of COPII-labelled ER exit sites from the periphery to the perinuclear region during interphase is also likely to rely on an interplay between the tER and microtubules [15]. In permeabilised cells tubulation of membranes caused by addition of Sar1-GTP is dependent on intact microtubules and also leads to recruitment of kinesin, the plus end-directed microtubule motor [57,58]. Conversely ER-to-Golgi transport of cargo-bearing intermediates relies on travel along microtubules in a minus-end-directed manner mediated by dynein [37]. Dynactin, a multi-subunit protein complex, interacts with both dynein and kinesin through a single subunit p150Glued [59]. The other subunits of the dynactin complex are thought to mediate interactions with cellular cargo such as organelles, or membrane-bound transport intermediates. Furthermore the p150Glued subunit is able to interact directly with microtubules, in particular the microtubule plus end, in the absence of a motor protein, which may enable a choice of motor to be made for a particular cellular cargo [60]. Following motor recruitment p150Glued is phosphorylated and the motor, e.g. dynein, would maintain the microtubule association. A recent study has found that p150Glued is able to interact with the COPII component Sec23 and shows some colocalisation in cells [32]. This suggests that Sar1-Sec23-p150Glued could mediate interaction between the tER and microtubules. As the localisation of p150Glued to microtubule plus ends does not seem to be necessary to mediate membrane transport [61], the COPII-dynactin interaction could facilitate the spatial stability of ER exit sites. How this would hand over to the dynein-mediated transport of COPI-coated Golgi-directed intermediates is a fascinating enigma. Microtubule tethering of ER exit sites is not necessary for their formation, as exit sites are maintained upon depolymerisation [27,32]. In the absence of microtubules the Golgi reversibly redistributes into mini-Golgi stacks through the cell utilising constitutive recycling pathways to the ER [55,62–64]. COPII-labelled puncta are found to cluster around these Golgi elements suggesting that their organisation may well be coupled to retrograde membrane traffic from the Golgi [27]. In steady-state cells retrograde transport from the Golgi can involve the formation of thin tubular processes that move out from the Golgi on microtubules in a kinesin-dependent manner [65,66]. This microtubule-dependent tubulation is exacerbated in BFAinduced Golgi redistribution [67,68] and the tubules are seen to 439 connect with ER exit sites [69] suggesting that these provide the target for fusion of the retrograde intermediates. Thus while the inter-relationship between ER exit sites and microtubules is well documented, there remains little mechanistic understanding of how the coupling of membranes to the microtubules is regulated. 3. COPII and cargo in living cells 3.1. COPII coat dynamics The use of GFP chimaeras of the COPII components has enabled close visualisation of the kinetics of membrane association of the individual proteins. The dynamics of coat assembly/disassembly is measurable through the biophysical properties of GFP. While GFP is generally a stable fluorophore that is resistant to photobleaching under standard imaging conditions, it is possible to completely photobleach GFP chimaeric proteins using a high intensity laser light directed to a specific region of interest, and this bleaching is irreversible [70,71]. Exchange of photobleached GFP molecules with unbleached GFP molecules from outside the region of interest is then measurable under normal low-intensity laser imaging conditions, known as fluorescence recovery after photobleaching (FRAP). This reveals the speed at which the chimaeras are able to move within the cell. All of the individual COPII subunits as GFP chimaeras have been found to exchange very fast between a membrane and cytosolic pool [15,29,31,32]. Original recovery rates of 20–30 s were reported [29,31], but by reducing the bleach area a far more rapid rate for recovery of less than 5 s was found [32,72]. It is therefore likely that COPII components undergo repeated cycling rounds in the same area rather than through exchange with distant ERES [72]. These cycling dynamics are slowed in the presence of GTP-restricted Sar1 and the immobile fraction (i.e. the protein that remains bound to the membrane) is increased [29,72]. Interestingly the FRAP dynamics of Sar1-YFP revealed that its turnover is approx fourfold faster than either Sec23 or Sec24 chimaeras [72]. Kinetic modelling of this binding difference suggests that COPII could remain bound transiently to membranes even after GTP hydrolysis and release of Sar1 into the cytosolic pool [72], as compared to the dogma whereby COPII would be completely released from the ERES by the activity of GTP hydrolysis. The behaviour of COPII would therefore be analogous to the role proposed for COPI, in which the interaction with effector molecules stabilises COPI on the membranes after GTP hydrolysis and release of Arf1 [34]. This provides further evidence that COPII might be envisaged to stabilise membrane domains rather than to promote vesicular assembly. 3.2. Cargo release alters COPII kinetics When protein synthesis is blocked with cycloheximide, COPII (Sec23) exchange increases at the tER, while the steadystate distribution of COPII associated with tER reduces [72]. Conversely the exchange rate of Sar1 on and off the membrane reduces while the amount associated with tER increases under 440 S.J. Kirk, T.H. Ward / Seminars in Cell & Developmental Biology 18 (2007) 435–447 the same conditions. This would suggest that, without cargo, Sec23 (and presumably the other heterodimer COPII subunits) is destabilised on the membrane. Similarly in cells expressing the cargo protein VSVG at the restrictive temperature, where VSVG is unable to fold and is retained in the ER, Sec23 exchange rates increase [72]. As VSVG is released from the ER upon incubation at the permissive temperature, COPII exchange rates reduce slightly but remain faster than at steady state. Sec23 can bind VSVG at nonpermissive temperature [73], therefore a possible explanation is that in vivo Sec23 is repeatedly interacting with the VSVG cytosolic tail, bringing the VSVG to the tER. However, an additional factor must be involved in dictating whether the VSVG is competent for transport, perhaps through its oligomerisation, which could cause clustering of associated Sec23 and might then drive self-assembly of COPII [74]. As the VSVG folds, the availability of a now folded, transport-competent, secretory cargo alters the COPII turnover kinetics but the cargo availability appears to maintain this above steady-state levels. Golgi enzymes can be restricted to the ER through BFA treatment, which prevents COPI membrane association and causes reversible disassembly of Golgi membranes [67,75]. Under these conditions the increased ER-based cargo causes a slowing in COPII exchange with a reduction in mobile fraction [29] which seems to contradict the response with VSVG. However, the COPII machinery interacts differently with alternative trapped cargoes, e.g. the GTP-restricted Sar1 block causes accumulation of VSVG or lumenal-GFP (an ER-directed GFP molecule that is usually rapidly secreted [48]) into the clustered membranes at the perinuclear region while Golgi enzymes, procollagen, or GPI-GFP are localised to the ER [49,63,64]. Therefore in BFA-treated cells where COPI can no longer bind to membranes, perhaps the slowed rate of COPII exchange may reflect an inability to facilitate the exit of the Golgi proteins through the usual downstream recruitment of COPI. 3.3. COPII kinetics and the lipid environment Lipids have been implicated in facilitating COPII activity, and ER export. Inhibition of phospholipase D-mediated phosphatidic acid (PA) formation releases COPII from the membrane, blocking cargo export [76], and prevents the formation of Sar1dependent tubules in permeabilised cells [58,76]. Furthermore, p125, a PA-specific phospholipase A1, alters COPII assembly when overexpressed or depleted [77,78], suggesting that modulation of the lipid layer may be required to support coat assembly. Depletion of sterols delays export of VSVG, a model transmembrane cargo protein, from the ER [42]. But although COPII distribution (Sar1 or Sec23) is not obviously affected under these conditions, Sec23 turnover slows by 40% while Sar1 kinetics are unchanged. This would suggest that the delay in cargo export would be caused by the reduced COPII coat efficiency. Kinetic modelling approaches applied to this system show that this corresponds to a slowed rate of GTP exchange on Sar1 leading to reduced recruitment of Sec23, together with a longer residence time for COPII on ERES [42]. How the GTP-exchange factor (GEF) for Sar1, Sec12, is modulated by altered sterols is unknown. 3.4. Cargo modulation of ERES organisation The effect of cargo egress on the number and distribution of ER exit sites has been researched in a number of approaches. EM quantitation of ER budding profiles found that release of VSVG from its conditional block caused an increase in budding profiles [73]. Interestingly incubation of the viral glycoprotein in the ER during the high temperature block causes a decrease in ER exit buds as compared to a mock-infected control suggesting that COPII may associate with the VSVG while it is held in the ER and an interaction between Sec23 and VSVG at restrictive temperature has been confirmed biochemically [73]. Efflux of VSVG increases both COPII membrane association and number of ER exit sites, presumably to accommodate the cargo wave [79]. Given the functional role of COPII in procollagen exit [41], but the unexplained positional difference [17], it would be very interesting to see if the behaviour of COPII is similarly modulated in cells synthesising procollagen, or indeed other large cargoes. In cells where neither COPII nor COPI can associate with membranes (through treatment with H89 and BFA together), proteins of the early secretory pathway become trapped in the ER [67,80,81]. Washout of cells with fresh media creates a wave of Golgi ‘cargo’ which also affects ERES dynamics. Compared to steady-state levels of COPII membrane binding (visualised with Sec13-GFP), the efflux of Golgi proteins causes an increase in COPII assembly on the membranes, both in terms of total protein bound, but also in numbers of ER exit sites, although the individual intensity of these sites does not appear to increase, i.e. cargo movement creates formation of larger ER exit sites but the COPII is not more concentrated [79]. ER-localised Golgi enzymes (by overexpression or BFA-induced redistribution) also induce upregulation of GTP-dependent COPII–membrane association which reduces to steady-state upon BFA washout [79]. This highlights the adaptive nature of the COPII cycle, where the cytosolic pool of COPII subunits enables a quick response to the requirement to sort high levels of cargo. 4. ER exit site biogenesis 4.1. Changes in ERES number during the cell cycle and self-organisation Transitional ER sites exhibit organised architecture through the cell and the mechanisms that generate this organisation are the subject of great research efforts. During mitosis in mammalian cells, COPII no longer associates with the membrane [15,27,82,83], leading to an arrest in anterograde transport [82]. ER exit sites are not visible in mammalian cells by either light or electron microscopy, although tiny buds on the surface of the ER are apparent during metaphase [82]. The loss of ER exit sites specifically occurs in metaphase, after microtubule breakdown, when COPII components are found dispersed throughout the cytosol and are not S.J. Kirk, T.H. Ward / Seminars in Cell & Developmental Biology 18 (2007) 435–447 membrane-associated [27,83]. As the cell progresses through mitosis, COPII components are seen to re-associate at discrete points, which soon after show colocalisation with Golgi proteins emerging from the ER [83]. What determines the timing of ER exit site nucleation, and the location, is unknown. As COPII is released from membranes after microtubule breakdown and rebinds prior to spindle disappearance, it seems unlikely that microtubules are directly responsible for COPII dynamics in this situation. Furthermore, during interphase, ER exit site numbers increase with cell size, such that immediately prior to prophase cells have double the number of ERES that they had at the start of interphase [27]. Peripheral sites increase in number by 50% over 4 h and these newly formed export sites slowly translocate to the juxtanuclear area (5–15 ␮m/h) [15]. During the de novo formation, there is a slow increase in fluorescence to a plateau equivalent to adjacent ERES while some ERES are seen to undergo either fusion or fission [15]. Newly formed or newly divided ERES rapidly acquire additional Sec23. The increase in numbers of ERES proportional to ER growth in interphase would ensure that sufficient ERES components are available to ensure appropriate segregation between daughter cells following cell division. Other model organisms also show the ability of ER exit sites to proliferate de novo. Live imaging of Sec13-GFP in P. pastoris has found that ERES can form de novo in the cytosol and that Golgi structures only appear at tER sites after the Sec13 localisation is already apparent [84], similarly in T. brucei [24]. In T. gondii the ER exit site has not been followed in time-lapse studies observing cell division [85], but it has been reported that fixed electron micrographs of cells at different cell cycle stages suggest that it undergoes elongation and medial fission, at the same time as the Golgi apparatus [86]. As there is no spatial separation between the exit sites in Toxoplasma it is unclear whether the same mechanisms may control this duplication, but it cannot be described as de novo in the same way as is apparent in other organisms. What controls the formation of these tER sites de novo is still poorly understood. One postulated mechanism is that ER exit sites are maintained by a self-organising principle of size control in which self-organisation of one or more components can enable formation of a structure de novo, e.g. the polymerisation of tubulin heterodimers to form microtubules [87,88]. In this model maintenance of existing tER sites would result from a balance between the budding of COPII ‘vesicles’ and the recruitment of new molecules at the edge of these sites [15,84]. Formation of new tER sites could arise by local concentrations of cargo-associated COPII proteins associating through weak affinities to nucleate a new specialised patch in the ER membrane [84]. 4.2. ‘Early’ COPII components with putative role in tER organisation The order of assembly of COPII was established by sequential addition of S. cerevisiae recombinant COPII components to an in vitro assay and subsequently confirmed in mammalian cells 441 [1,43,89,90]. However, other COPII components were found to be required for the budding reaction that may play a role in organising ER exit sites as described below. Much of the clarification of the role of these putative upstream components has relied on the use of Sar1 mutants. As Sar1 is a member of the Ras family of small GTPases its activity is reliant on a conformational switch dependent on which nucleotide is bound. When GTP bound, Sar1 is active on the membrane and can recruit downstream effectors, and when GDP bound it is inactive and cytosolic. Manipulation of Sar1 activity can be achieved by the expression of dominant mutant forms of Sar1, H79G (or equivalent amino acid substitution in different organisms) for the activated GTP-bound Sar1, and T39N (or equivalent) for inactive GDP-bound Sar1 [90]. 4.2.1. Sec12 Mammalian Sec12 is a type II transmembrane protein with a large cytosolic domain that promotes Sar1 activation, i.e. it is the GTPase-specific guanine nucleotide exchange factor (GEF) [91]. It is found localised with VSVG at 40 ◦ C but no longer shows an overlapping distribution when VSVG is held at ER exit sites by a 15 ◦ C block. Therefore it appears that at steadystate Sec12 is distributed throughout the ER membranes [91]. A similar distribution has been reported for a GFP-tagged mSec12 chimaera, which shows free diffusion through ER membranes [15]. While S. cerevisiae Sec12p is also localised to the ER [92], in sharp contrast P. pastoris Sec12 is found localised to tER puncta [20] but it is freely mobile and can exchange with the ER pool [93]. Overexpression of Sec12 in Pichia causes localisation to the ER but does not affect downstream COPII components or Golgi localisation [93]. There is a possibility that mammalian Sec12 is found at the tER but this may be masked either by antibody artefact, or by the GFP chimaera expression levels potentially causing an overexpression phenotype similar to Pichia. Interestingly, Sec12 RNAi does not cause obvious disruption to ERES organisation, with no change in number and distribution, though the fluorescence of Sec23 localised to ERES is less intense [94]. Conversely Sec12 RNAi does prevent the reaccumulation of Golgi enzymes in the perinuclear region following BFA washout [94]. Therefore, as compared to the lack of effect on ERES organisation that could be attributable to incomplete Sec12 degradation, this demonstrates that RNAi depletion is at significant levels to have an effect on Sec12 function. This shows that Sec12 is required for COPII-regulated exit from the ER and when the Sec12 is depleted cargo molecules, in this case Golgi enzymes, remain trapped in the ER. Therefore while it appears that Sec12 is needed for cargo transport out of the ER, it is not required for organisation of the ER exit sites. The fact that localisation of downstream COPII components to tER is not inhibited upon Sec12 depletion by RNAi suggests that the Sec23/24 heterodimer can assemble on the membrane without Sar1 activation, maybe through interaction with cargo molecules in the ER. This has also been shown in Pichia pastoris through expression of Sar1T34N, the GDP-bound Sar1 mutant, where secretion is inhibited but Sec13 and Sec12 are still able to localise at punctate tER structures [95]. 442 S.J. Kirk, T.H. Ward / Seminars in Cell & Developmental Biology 18 (2007) 435–447 4.2.2. Sec16 Recently another upstream coat component, Sec16, has become the focus of interest in tER organisation. Sec16 was characterised in S. cerevisiae as a large hydrophilic protein tightly bound to ER membranes [96]. Further work described interactions between Sec16 and various COPII components including Sec23, Sec24, Sec31 [97,98], and found that interactions between Sec16 and Sar1-GTP might facilitate COPII assembly (although Sec16 is not absolutely required for this process) and also prevent the COPII coat from disassembling prematurely during Sar1-dependent GTP hydrolysis, suggesting a role for Sec16 in ER exit nucleation [99]. Investigation of tER assembly in P. pastoris found that in a mutant Sec16 strain, the tER localisation of Sec12 is disrupted and it is now found in an ER distribution, while Sec13-GFP remains in punctate structures that are more numerous and smaller than the wild type morphology [95], resembling that found in S. cerevisiae. Furthermore the Golgi stacks also more closely resemble single disparate cisternae as found in the budding yeast with early and late Golgi markers no longer aligned [95]. Therefore it appears that in this organism Sec16 has a direct effect on tER assembly and organisation [95]. A speculative model for how Sec16 might organise tER sites in Pichia proposes that as Sec16 is substoichiometric to other COPII subunits, it might bind at the edge of COPII clusters [84] and that through self-association it could crosslink the clusters to create multimeric assemblies [95]. Recently mammalian Sec16L has been identified in three parallel studies [94,100,101]. A second smaller homologue Sec16S was also identified that seems to confer a nonredundant function analogous to the larger protein [94]. Sec16 localises with other COPII components at tER puncta with some ER background. A similar distribution is found for GFP-Sec16L. FRAP of the protein in tER puncta leads to a variable recovery rate (t1/2 recovery = 4–27 s) as compared to other COPII subunits which have relatively consistent recovery rates [32,72,100]. The basis for this variability is unknown. Overexpression of Sec16 chimaeras causes loss of the punctate localisation of Sec23/Sec24 or Sec13/Sec31 heterodimers [94,100,101], a reduction of peripheral puncta that label for early secretory components such as ERGIC-53 or ␤-COP [100,101], and an inhibition of VSVG exit from the ER [100]. While Golgi proteins maintain localisation to the perinuclear region, the Golgi morphology appears altered [94,100,101]. The results looking at depletion of Sec16L with RNAi are slightly variable between the three studies, though essentially depletion of Sec16 causes a reduction in tER sites and redistribution of ERGIC-53 to the ER [94,100,101]. There is a reduction in peripheral spots labelled for ␤-COP but the Golgi pool is retained [100], and Golgi proteins maintain a juxtanuclear position, albeit slightly altered compared to unaffected cells, although the Golgi ultrastructure through EM has not been examined to look at cisternal integrity [94,100]. Sec16 depletion markedly reduces cargo export as measured by either VSVG transport to the plasma membrane [100,101], or by visualising export of a Golgi protein following BFA washout [94]. Therefore it appears that in mammalian cells Sec16 has an essential role in tER organisation and cargo export. 4.2.3. Cooperation of Sec16 and Sar1 in mammalian cells The effect of Sar1-GDP in mammalian cells causes almost total loss of identifiable tER structures labelled for Sec16, or later COPII components (Sec23/24, Sec13/31) [29,100,102], while Sar1-GTP causes accumulation of these components in a juxtanuclear region [29,49,56,100,101] although under these conditions Sec16 is found on the juxtanuclear cluster as well as on reduced numbers of peripheral puncta that are not labelled for later COPII components [100]. It has been proposed that COPII coat proteins are associated with free vesicles and tubules [19,56] and that as COPII-coated ERES can exhibit a slow centripetal movement towards the MTOC [15], late COPII-labelled membranes may cluster to the MTOC under the effect of Sar1-GTP [56] while the Sec16 structures would be immobile in the cell periphery [100]. However, as the juxtanuclear membrane clusters show continuity with the ER [29], also label for Sec16, and late COPII components are not found at the peripheral puncta [61] further work is needed to explain this effect. Sar1 had been proposed to link cargo selection to tER assembly [58] but the lack of tER sites in S. cerevisiae and the inability of Sar1-GDP to disrupt tER sites in P. pastoris suggested that it may not be a contributing factor in nucleation of ERES. While Sec16 appears to be a major tER determinant in Pichia it appears from the application of Sar1-GDP that the role of Sar1 may be more significant in mammalian cells. However, when GFP-Sec16 is overexpressed in the presence of GDP-restricted Sar1, it localises to peripheral punctate structures, but Sec24 is unable to associate with these structures. This suggests that Sec16 requires Sar1 activity for ERES localisation at endogenous levels, potentially to prevent its dissociation, but when overexpressed YFP-Sec16 can assemble at ERES independent of Sar1-GTP loading [100]. It appears that upon overexpression Sec16 can self-associate to form an assembled site so that ERES are maintained even in the presence of Sar1-T39N. Furthermore overexpression of YFP-Sec16 with GTP-bound Sar1 causes accumulation of Sar1 and Sec16 together in large curved structures that do not label for late COPII subunits and are not clustered at the MTOC [100]. While overexpression could be argued to create artefacts, these data would suggest the possibility of self-assembly of higher order Sec16 structures in mammalian cells to nucleate or demarcate ER exit sites, but that Sar1 is additionally required for the organisation and maintenance of tER sites. It has been postulated that GTP hydrolysis could cause Sar1 to be released from assembled COPII structures, leaving a stabilised polymeric coat on the membrane. Therefore Sar1GTP may be excluded from the central area of the coat, but it could be recruited to stabilise the coat perimeter [103], analogous to the role proposed for Sec16 in Pichia [95]. This suggests that in mammalian cells the two models could be combined such that Sec16 and Sar1 cooperatively maintain the tER sites. 4.2.4. p125 A further candidate for tER nucleation is p125, a mammalianspecific peripheral membrane protein with phospholipase A homology that binds Sec23 at the tER [77]. Interestingly overexpression of p125 causes ERES clustering particularly in the perinuclear region with downstream defects in ERGIC and Golgi S.J. Kirk, T.H. Ward / Seminars in Cell & Developmental Biology 18 (2007) 435–447 protein localisation [77,78], and if p125 is redistributed to the cytosol COPII membrane binding is diminished [78]. p125 depletion results in loss of perinuclear accumulation of ERES but peripheral sites appear unaffected. Cargo transport is also unaffected. So p125 might be involved in ERES architecture but not their functionality. Overexpressed Sec16 causes depletion of p125 from ERES as might be expected for a downstream COPII effector. However, intriguingly, unlike Sar1-GTP accumulation in juxtanuclear membranes, p125 overexpression causes accumulation of later COPII subunits, but not Sec16, which loses its steady-state peri-Golgi concentration and is found only on peripheral structures [101]. p125 may therefore affect membrane dynamics by linking the membrane lipids through its phospholipase A domain with the COPII late subunits. 4.2.5. p115 Another postulated ER exit site determinant is p115 where p115 depletion in Drosophila S2 cells shows disruption of tER puncta labelled for Sec23 while there is little disruption to overall secretory cargo flux [104]. p115 is incorporated into COPII vesicles in vitro [105] but it is not clear that this protein is acting at the level of COPII organisation per se, as its functions have been implicated at later trafficking steps [106]. However, it may provide a link between COPII and COPI handover, as additional evidence suggests that in the presence of GTP-bound Sar1, where COPI is unable to associate with cell membranes [29], the juxtanuclear concentration of ER exit sites labelled for Sec13 is intriguingly dependent on p115 [56]. Further putative ERES determinants include PCTAIRE-1, a PCTAIRE kinase of the cyclin-dependent kinase family, that binds Sec23 and when deficient causes mild phenotypic aberration in tER localisation but does not affect cargo trafficking [107], and Nm23H2 [108], an isoform of nucleoside diphosphate kinase, that alters both ERES distribution and inhibits cargo trafficking when depleted, potentially paralleling the role of Nm23H1 in endocytosis [109]. 4.3. The role of COPII in Golgi biogenesis and maintenance To what extent the activity of COPII in ER exit modulates Golgi membranes has been the subject of great controversy [18,110,111]. The debate hinges over whether the Golgi acts as an autonomous organelle, or whether it is dependent on cycling pathways to and from the ER. This is particularly critical during mitosis, as one school of thought argues that recycling to, then retention of, Golgi proteins in the ER during mitosis facilitates distribution of the components between the two daughter cells [64,83]. In lower eukaryotic cell division the mechanism of ER and Golgi division between daughter cells seems to depend on functional ER exit. Utilising S. cerevisiae strains where ER inheritance is compromised (e.g. sec8-599), it was found that by preventing ER from entering the daughter bud, Golgi membranes did not appear in the nascent bud [112]. As COPII is usually present in very small buds [20] this implies that by preventing ER entering the bud, ER exit is unable to occur there, 443 and new Golgi stacks are unable to form. In the yeast P. pastoris and the protozoan T. brucei newly formed Golgi stacks appear at the same time as newly formed tER sites, implicating a role for the tER in generating the Golgi [24,84]. Microinjection of the Sar1-H79G mutant (GTP-bound form; [90]) into mammalian cells causes accumulation of Golgi proteins in the ER over the course of 2 h [29,64,113]. This reflects the constitutive recycling of Golgi proteins to the ER, but with a block in ER exit incurred by the Sar1 mutant, the Golgi proteins are unable to return, and therefore are retained in the ER. When this is repeated in cells that are about to undergo mitosis, Golgi proteins are unable to re-associate into characteristic juxtanuclear membranes as daughter cells are formed [64]. This suggests that Golgi proteins are dependent on Sar1 to reform a Golgi structure following cell division, indicating that they are trapped in the ER during mitosis and are unable to exit in a Sar1 mutant block [64]. More recently co-imaging of Sec13-YFP with GalT-CFP to mark COPII and Golgi, respectively revealed that assembly of the COPII coat was seen first in mitotic cells progressing through anaphase, and only in telophase were Golgi proteins then seen to begin to exit the ER, appearing closely associated with Sec13-labelled puncta [83]. Furthermore highresolution imaging has clearly shown the localisation of Golgi proteins to strands of the ER during metaphase [83]. Conversely the use of Sar1 mutants has also been used to describe the non-ER vesicular intermediate for the Golgi in mitosis [110,114,115] whereby the block of brefeldin A was used to trap Golgi enzymes in the ER and block COPI-dependent ER exit, but the subsequent injection of GTP-restricted Sar1 and BFA washout enabled accumulation of a subset of Golgi components to localise to the perinuclear region in a Golgi-like assembly as cells exit mitosis. As the tER is colocalised to the same region, this may reflect a Sar1-dependent redistribution of the tER, as discussed above, rather than a Golgi reassembly [29]. However, there is some dispute as to whether the Golgi matrix proteins under these conditions are labelling the same membranes as the COPII subunits [56]. To mimic the processes in mitotic cells, and to enable a breakdown of events occurring at ERES, the PKA (protein kinase A) inhibitor H89 has been found to redistribute COPII components from the ERES to the cytosol [81] while Golgi protein recycling pathways are blocked, or greatly slowed, by immobilisation of Arf1 on the membrane [81,116]. In vitro H89 inhibits Sar1 and Sec23/24 membrane binding and thereby prevents cargo export [80]. The target for H89 inhibitory effects on ER export and COPII assembly does not appear to be PKA [81,117], but the kinases identified through other approaches (e.g. PCTAIRE-1 or Nm23H2 above [107,108]) may be the potential H89-sensitive factors. It may therefore be necessary for the ER export machinery to interact with kinase signalling cascades to exact cargo selection [80]. Golgi enzymes relocate to the ER by the action of BFA [67] and can then reform a Golgi upon BFA washout. Golgi matrix proteins accumulate at ER exit sites in the presence of BFA [29] but become diffusely localised if H89 is added [118]. Washout of both reagents enables reassembly of all the Golgi components in the juxtanuclear region [118]. Emergence of cis-Golgi matrix pro- 444 S.J. Kirk, T.H. Ward / Seminars in Cell & Developmental Biology 18 (2007) 435–447 teins precedes other Golgi components [79,118,119] and may reflect the presence of sorting signals that enable interactions (potentially by direct binding) with the COPII complex. If the washout occurs in the presence of nocodazole, which prevents repolymerisation of microtubules, clearance of Golgi proteins from the ER into mini-stacks is faster [119]. In late telophase GM130 and giantin appear faster into puncta alongside COPIIlabelled structures than Golgi enzymes [119], i.e. H89 washout resembles functional behaviour. Comparison of cycling kinetics of Golgi enzymes in nocodazole ministacks and in telophase, shows that the kinetics of Golgi enzyme exchange is faster in mitotic cells than even in nocodazole treatment, suggesting that the membranes have not formed into organised stacks and may maintain closer interaction with COPII until the microtubule array regrows, and the Golgi reforms next to the nucleus during cytokinesis [83]. 5. Concluding remarks What is particularly striking is that despite the temporal stability of ER exit sites, they are dynamic structures that can rapidly reorganise in response to trafficking perturbants or during cell morphogenesis. During muscle differentiation, for example, diffusely distributed COPII puncta become aggregated around nuclei, closely linked to a redistribution of microtubules to multiple nucleation sites [120]. The inter-relationship between the tER and microtubules seems to underly tER organisation, and how tER nucleation is regulated in the absence of microtubules is an interesting enigma, yet while our understanding is improving much remains unanswered. Furthermore it is clear that the interaction between COPII components and membrane lipids is proving essential to the formation and function of COPII-dependent sorting and transport processes. Not only are they required for recruitment fidelity of COPII components [42,80], but they appear necessary for the maintenance of tER structures [101] and the implication that kinase signalling pathways could be directed to lipid modification or recruitment at ER exit sites underscores this possibility. Recent evidence using a permeabilised cell system suggests that specific lipids are required for the formation of cargo export domains which would create a lipid environment in the ER exit sites that is different from the adjoining ER membranes [121]. 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Glossary Arf1: small GTPase that recruits COPI coat complex BFA: brefeldin A; inhibits COPI binding to membranes through inactivation of Arf1 and causes Golgi membranes to fuse back into the ER EM: electron microscopy ERES: ER exit site; comprises COPII- and COPI-coated membranes ERGIC: ER–Golgi intermediate compartment; equivalent to VTC 447 FRAP: fluorescence recovery after photobleaching; a technique utilizing the irreversible photobleaching properties of GFP to visualise movement of chimaeric proteins within living cells. GEF: guanine nucleotide exchange factor; catalyses exchange of GDP for GTP on small GTPase (Sec12 is the Sar1 GEF) GFP: green fluorescent protein GPI: glycosyl phosphatidylinositol; lipid anchor found on some plasma membrane proteins H89: a protein kinase A inhibitor that prevents COPII binding to membranes MTOC: microtubule organising centre Nocodazole: prevents polymerisation of microtubules tER: transitional ER; COPII-labelled budding region of ER VSVG: vesicular stomatitis virus glycoprotein; transmembrane cargo protein with a temperature sensitive mutant form that reversibly misfolds in the ER at high temperature, and correctly folds and traverses the secretory pathway when temperature is dropped VTC: vesicular–tubular cluster; COPI-labelled membranes closely associated with transitional ER