CELB40060 Membrane Trafficking in Animal Cells. Prof. Jeremy C. Simpson. Lecture 2 COPII and export from the ER

Transcription

1 CELB40060 Membrane Trafficking in Animal Cells Prof. Jeremy C. Simpson Lecture 2 COPII and export from the ER

2 Today s lecture... The COPII coat - localisation and subunits Formation of the COPII coat at ER exit sites (ERES) Budding of COPII-coated carriers ER-to-Golgi transport Signals for ER export

3 Coat protein complexes * retromer COPII exit from the endoplasmic reticulum COPI return to the endoplasmic reticulum, integrity of the Golgi complex clathrin* exit from the TGN, recycling to the TGN, endocytosis from the cell surface retromer endosome to TGN * the specificity of the clathrin coat is determined by a family of adaptor proteins

4 Localisation of the COPII coat - the COPII coat was originally identified as being a 10nm thick proteinaceous coat found on 60-65nm vesicular profiles - it was seen to be clearly different from the previously identified clathrin coat and COPI (coatomer) coats Barlowe et al. (1994) Cell 77: COPII: A membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. - mutations in components of the COPII coat can lead to changes in endoplasmic reticulum morphology and ultimately human disease - the rare genetic disease cranio-lenticulo-sutural dysplasia (CLSD) is associated with a mutation in the SEC23A gene of the COPII coat

5 Localisation of the COPII coat - in animal cells, transport carriers originating in the endoplasmic reticulum form at specialised sites termed the transitional ER (ter) or ER exit sites (ERES) - ERES are defined by the presence of subunits of the COPII coat complex - ERES are localised directly on the membranes of the ER, and in live cell imaging experiments move with the ER Hughes & Stephens (2008) Histochemistry and Cell Biology 129: Assembly, organization, and function of the COPII coat

6 Localisation of the COPII coat - in animal cells ERES appear to be relatively immobile, moving only small distances within the cell - this implies that either the COPII coat does not remain on the transport carrier as it moves from the ER to the Golgi complex The Sec24D subunit of COPII tagged with YFP Wessels & Simpson (2007) Seminars in Cell and Developmental Biology 18: Impact of live cell imaging on coated vesicle research.

7 Localisation of the COPII coat - in animal cells ERES are distributed throughout the cytoplasm, but are also seen to cluster in a juxta-nuclear position close to the Golgi complex - ERES are able to undergo fission and fusion events, and can also form de novo - COPII-coated membranes are often localised very close to COPI-coated membranes, implying a sequential role for these two coat complexes in transport between the ER and Golgi complex 10 µm COPII coat (ERES) COPI coat (VTCs and Golgi)

8 Subunits of the COPII coat - the COPII coat complex is composed of 5 core COPII components - the small GTPase Sar1 - the heterodimeric complex Sec23/Sec24 - the heterotetrameric complex Sec13/Sec31 - in mammalian cells a number of subunit isoforms exist - Sar1A and Sar1B - Sec23A, Sec23B, Sec24A, Sec24B, Sec24C, Sec24D - Sec13, Sec31A, Sec31B - the ultrastructure of COPII-coated vesicles was only solved in 2007 by the lab of Jonathan Goldberg Sec13 Sec31 Fath et al. (2007) Cell 129: Structure and organization of coat proteins in the COPII cage Sar1 Sec23 Sec24

9 Formation of COPII-coated buds at ERES - COPII buds are initiated by the activation of the small GTPase Sar1, as a result of recruitment by the guanine nucleotide exchange factor (GEF) Sec12, a type II transmembrane protein - upon exchange of GDP for GTP, Sar1 exposes an N-terminal amphipathic tail that embeds in the membrane - Sec12 appears to be important in maintaining the supply of Sar1-GTP on the membrane, in turn enhancing cargo capture by COPII The small GTPase cycle - in GDP-bound state the GTPase is inactive (eg: bound to a guanine dissociation inhibitor GDI) - a guanine nucleotide exchange factor (GEF) replaces the GDP with GTP - in GTP-bound state the GTPase is active and can recruit effectors - the GTP is hydrolysed back to GDP by a GTPase activating protein (GAP) - the GTPase is available for another round of activity

10 Formation of COPII-coated buds at ERES - Sar1-GTP recruits two components of the COPII complex (Sec23 and Sec24) from the cytoplasm - Sec23 acts as a GTPase activating protein (GAP) for Sar1 - Sec24 binds the cytoplasmic tails of certain cargo receptor proteins - together Sar1, Sec23, and Sec24 form the prebudding complex - the bow-tie shape of Sec23/24 induces curvature of the ER membrane

11 Budding of COPII-coated carriers - the outer shell of the COPII coat (Sec13 and Sec31 proteins) is then recruited from the cytoplasm - the cumulative addition of Sec23/24 and Sec13/31 induces further curvature in the ER membrane, forming a bud, and ultimately a vesicle - the Sec13/31 subunits have a structural role in COPII cage formation - the COPII-coated vesicle buds from the ER, but appears to remain attached to the microtubule cytoskeleton Watson et al. (2005) Nature Cell Biology 7: Coupling of ER exit to microtubules through direct interaction of COPII with dynactin.

12 How is an ERES defined? - when Sec12 is delocalised into the general ER, ERES remain intact - this implies that Sec12 itself does not define thee site of COPII recruitment - the original secretory pathway screen in yeast identified the gene SEC16 as being important for cargo exit from the ER - recent functional experiments have showed that either increased or decreased levels of Sec16 protein influence the distribution of ERES - overexpression of Sec16 causes a loss of the COPII subunit Sec24 in ERES - downregulation of Sec16 causes a reduction in the number of distinct COPIIcoated structures Watson et al. (2006) Traffic 7: Sec16 defines endoplasmic reticulum exit sites and is required for secretory cargo export in mammalian cells.

13 How is an ERES defined? Watson et al. (2006) Traffic 7: Sec16 defines endoplasmic reticulum exit sites and is required for secretory cargo export in mammalian cells.

14 How is COPII vesicle formation regulated? - the cell must ensure that COPII vesicles only form when cargo is ready for transport - of particular importance is the assembly of transmembrane cargo and transmembrane cargo receptors in the ER membrane (for example ERGIC53) - in vitro experiments using liposomes indicate that continual recruitment of Sec23/24 to the ER is maintained by the action of the GEF Sec12 - the interaction of Sec23/24 with transmembrane cargo appears to stabilise the prebudding complex - this effectively acts as a timer, allowing further Sec23/24 to be recruited, therefore overcoming the rate of GAP activity of Sec23 - through its hydrolysis therefore, Sar1 appears to selectively prevent the formation of empty COPII vesicles

15 How is COPII vesicle formation regulated? Sato and Nakano. (2007) FEBS Letters 581: Traffic 7: Mechanisms of COPII vesicle formation and protein sorting.

16 ER-to-Golgi transport

17 ER-to-Golgi transport COPII vesicles bud from the endoplasmic reticulum vesicles fuse with one another forming VTCs (vesicular tubular clusters) VTCs mature into the cis-golgi network (CGN) / intermediate compartment (IC) the COPII coat is rapidly disassembled from the vesicle VTCs move towards the Golgi complex, and become coated with the COPI coat

18 Signals for ER export - transmembrane proteins contain specific sequences in their cytosolic domains that act as export signals - these cytosolic tails interact directly with COPII subunits, in particular Sec24 - export signals are diverse, but many are composed of di-acidic motifs (D/E X D/E), short hydrophobic motifs (FF, IL,LL) or di-basic motifs (R/K X R/K)

19 Key take home point The COPII coat and its associated regulatory machinery ensures that cargo is selectively packaged into buds at specific sites on the ER (ERES)