Lecture 5 - Eukaryotic Membrane Transport 2024 PDF

LECTURE 5 – SYNTHESIS, TURNOVER AND TARGETING OF MEMBRANES Prof. Vassiliy BAVRO LIPID BIOGENESIS IN EUKARYOTES LIPID TRANSPORT – VESICULAR AND NON-VESICULAR CELLULAR POSTCODE AND GATED POST TRANSLATIONAL PROTEIN TRANSPORT: NUCLEAR...

LECTURE 5 – SYNTHESIS, TURNOVER AND TARGETING OF MEMBRANES Prof. Vassiliy BAVRO LIPID BIOGENESIS IN EUKARYOTES LIPID TRANSPORT – VESICULAR AND NON-VESICULAR CELLULAR POSTCODE AND GATED POST TRANSLATIONAL PROTEIN TRANSPORT: NUCLEAR IMPORT/EXPORT COMPARTMENTALISATION AND TRANSMEMBRANE TRANSLOCATION - ER-TARGETTING COATED VESICLES - CLATHRIN AND COPS PROTEIN VESICULAR FUSION AND TARGETING –SNARES and Rabs AUTOPHAGY (time permitting) ESCRT system (by Yourself!) As always, a star denotes an additional information slide for those curious and awake! CELLULAR MEMBRANE SYSTEMS ARE INTERCONNECTED! Approx 50% of the cell volume is taken up by membrane bound organelles  LIPID SYNTHESIS AND MEMBRANE BIOGENESIS IN EUKARYOTES - OVERVIEW 1. Majority of the lipid biosynthesis takes place in the ER Synthesis 2. Acyl-CoA shuttles between compartments. 3. Long-chain fatty acids are broken up in the peroxisomes. Acyl-CoA shuttle 4. Mitochondria burn the shorter chain fatty acids to CO2 and H2O in oxidative cycle. Degradation 5. Majority of the lipid membranes are transported as membrane vesicle precursors. J Lipid Res. 2009 Apr; 50(Suppl): S311–S316.doi: 10.1194/jlr.R800049-JLR200  LIPID SYNTHESIS AND MEMBRANE BIOGENESIS IN EUKARYOTES – NON-VESICULAR TRANSFER Lipid transfer proteins (LTPs) help transfer lipids via soluble intermediary LTPs form a soluble cage around the lipid (Think MlaC!) Membrane contact site (MCS) facilitate lipid transfer. In some cases, LTPs can contact both membranes Highly recommended review: doi: 10.1101/cshperspect.a013300  LIPID SYNTHESIS AND MEMBRANE BIOGENESIS IN EUKARYOTES – NON-VESICULAR TRANSFER CHOLESTEROL and PHOSPHOLIPID TRANSFER Translocation of nonesterified cholesterol (Ch) from Front. Cell. Infect. Microbiol., 19 September 2018 | https://doi.org/10.3389/fcimb.2018.00315 one membrane compartment to another is required for metabolic processing and membrane biogenesis refashioning. Two main groups of Ch-transfer proteins: StAR (Steroidogenic Acute Regulatory protein), family transfer proteins – mitochondrial linkage; Sterol Carrier Protein-2 (SCP-2) family. Unlike the StAR proteins, SCP-2 have broad specificity, facilitating the movement of various A. Cytosolic LTPs (e.g., STARD containing phospholipids and fatty acids in addition to Ch and is proteins 1-7) contain just a lipid-transfer also referred to as a nonspecific lipid transfer protein. domain (LTD) (green) but lack any membrane binding domain. Spontaneous intermembrane transfer of B. Lipid transport at MCSs by membrane phospholipids also occurs, but generally much more anchored LTP (e.g., ceramide transfer protein, slowly than Ch-transfer. CERT; oxysterol-binding protein, ORP3). MCS = Membrane Contact Sites  PROTEIN TRANSPORT BETWEEN COMPARTMENTS GATED Proteins can move from one compartment in cell to another by 3 main processes: Transmembrane transport – using membrane bound translocators to transport proteins across membranes. E.g. ER-transmembrane transport Gated transport – between cytosol and nucleus through nuclear pore complexes (NPCs). Vesicular transport – carry in vesicles. Figure 12-6 Molecular Biology of the Cell (© Garland Science 2008)  Nuclear Localisation Signal (NLS) Newly synthesised proteins get a destination tag - the signal sequence! Table 12-3 Molecular Biology of the Cell (© Garland Science 2008) The targeting signal for nuclear import is a poly-Lys signal The targeting signal for nuclear export has alternating bulky hydrophobics The targeting signal for the ER and export out of the cell contains a stretch of hydrophobic residues with a high alpha-helical propensity. Nuclear import/export is an example of folded protein transport AND it is also a GATED transport!  Nuclear pore – GATED TRANSPORT Unlike other transport mechanisms the proteins do NOT need to be unfolded, therefore only a single subunit of a multimeric complex needs a NLS. 500 macromolecules/sec are controlled through the NPC in both directions Figure 12-9 Molecular Biology of the Cell (© Garland Science 2008)  Nuclear pore complex (NPC) A fully assembled human NPC has an estimated molecular mass of ∼125 Mda, ~1,000 protein subunits (Reichelt et al., 1990), making it one of the largest protein complexes in the cell. Only about 30 different proteins – called nucleoporins (NUPs), but in multiple copies. Recent Advances! Science 15 Apr 2016:Vol. 352, Issue 6283, DOI: 10.1126/science.aaf1015  Nuclear pore complex (NPC) nucleoporins (NUPs) Nuclear pore complexes (NPCs) form a selective filter that allows the rapid passage of transport factors (TFs). TFs bind to the cargoes via the nuclear localisation signals (NLS) on the cargo. Intrinsically disordered proteins (IDPs) containing phenylalanyl-glycyl (FG)-rich repeats line the pore and interact with TFs. Hough et al. eLife 2015;4:e10027. DOI: 10.7554/eLife.10027  Nuclear Localisation Signal (NLS) NLS nuclear IMPORT pathway NES nuclear EXPORT pathway RanGAP (GTPase activator proteins in the cytoplasm): Activate the GTPase activity of the Ran resulting in GTP to GDP+Pi hydrolysis NTF2 – nuclear transport factor 2 – Allows transport of RanGDP into the nucleus RanGEF (GTP exchange factors) – in the nucleus: exchange the GDP for GTP ARPs = ankyrin repeat proteins travel directly with Ran (not importin dependent) The Small GTPase (G-protein) Ran coordinates the transport cycle. Its different conformations result alternatively in preference in binding to either importin or exportin cargoes. The nucleotide binding state of Ran depends on whether it is located in the nucleus (Ran-GTP) or the cytoplasm (Ran- GDP). Importin with cargo enters the nucleus by itself, but once inside the nucleus, interaction with Ran-GTP causes a conformational change in the importin that causes it to release its cargo. Protein translocation in the Endoplasmic Reticulum (ER) SCIENCE IS NOT SETTLED! SURPRISES STILL AWAIT! Completely novel insertion mechanism discovered in the ER membrane! 14th Dec 2017… EMC (ER membrane protein complex). Dec 2020! McDowell et al., 2020, Molecular Cell 80, 72–86 https://doi.org/10.1016/j.molcel.2020.08.012  Protein translocation in the Endoplasmic Reticulum (ER) Nascent polypeptide- associated complex (NAC) CO-TRANSLATIONAL: Guided entry of tail-anchored proteins (GET) pathway ER POST-TRANSLATIONAL insertases belong to the POST-TRANSLATIONAL: YidC/Oxa1/Alb3 family! chaperone EMC =Endoplasmic reticulum membrane protein complex CHECK THE HIDDEN SLIDES INCLUDED Hegde, R.S., Keenan, R.J. The mechanisms of integral membrane protein biogenesis. IN THE EXTENDED MOODLE VERSION Nat Rev Mol Cell Biol (2021).https://doi.org/10.1038/s41580-021-00413-2 FOR MORE DETAILS!  Multipass membrane proteins biogenesis in eukaryotes The PAT Complex - The PAT complex (Asterix + CCDC47 chaperones) engages and protects nascent TMDs with hydrophilic residues until they are buried in the protein interior upon folding. Substrates that are not shielded are potential targets for quality control (QC). PAT complex shielding could occur regardless of the route of TMD insertion. Hegde, R.S., Keenan, R.J. The mechanisms of integral membrane protein biogenesis. Nat Rev Mol Cell Biol (2021). https://doi.org/10.1038/s41580-021-00413-2  Multipass membrane proteins biogenesis in eukaryotes The “first draft” of ABCG2 topology is almost entirely wrong! It is being reordered by ATP13A1! A multipass membrane protein that initially inserts into the endoplasmic reticulum in a mostly inverted topology is post-translationally dislocated, re-inserted, and folded with the help of ATP13A1, a P-type ATPase. Co-translational translocation of the GABAA receptor subunit ends before the final TMD is inserted. This topologically incomplete product requires a rectification step that is mediated by a TMD insertase EMC. Ji et al., An ATP13A1-assisted topogenesis pathway for folding multi-spanning membrane proteins. Molecular Cell, Volume 84, Issue 10, 16 May 2024, Pages 1917-1931.e15 https://doi.org/10.1016/j.molcel.2024.04.024 Wu et al., EMC rectifies the topology of multipass membrane proteins. Nature Structural & Molecular Biology volume 31, pages32–41 (2024) https://doi.org/10.1038/s41594-023-01120-6  VESICULAR TRANSPORT Transport between compartments that are topologically identical occurs by vesicle transport Molecules can be moved from the ER to the Golgi and then cell surface, and at the time of fusion the contents of the vesicle are expelled extracellularly Figure 12-5&7 Molecular Biology of the Cell (© Garland Science 2008)  VESICLES AND MEMBRANE GENERATION MOST MEMBRANES TRAVEL AS COATED VESICLES! Coat protein II (COPII; green) forms vesicles that transport from the ER to the Golgi. COPI (purple), forms vesicles for transport in the other direction, from the Golgi to the ER. Clathrin (blue) is associated with both Golgi-to-endosomal and plasma-to-endosomal transport, but uses different adaptor proteins (APs) – AP1 and AP2 respectively, plus AP3 for Golgi-to-lysosome. Hsu et al., 2009 Nature Reviews Molecular Cell Biology 10, 360-364 (2009).  COATED VESICLES retrograde clathrin clathrin clathrin anterograde Sar1 Arf1 PIP2 – a lipid (Arf1 in AP1?) Schematic view of the secretory pathway and representation of the major coat proteins that mediate protein sorting at different cellular compartments. Gomez-Navarro et al., 2016, 215 (6):279 DOI: 10.1083/jcb.201610031  COATED VESICLES Clathrin, COPII and Coats Clathrin COPI skeletons to scale AP1/2/3 bind cargo. No small GTPase involved! Adaptors Signalling lipid – phosphatidyl-inositol phosphate (PIP), More precisely PI(4.5)-bisphosphate aka PIP2 COP II – anterograde (forward) Sec23/24 bind cargo and/or SNAREs and interface with Sar1 GTPase COP I – retrograde; Ret3 binds cargo proteins; Sec21/26 interface the Arf1 GTPase Trends in Cell Biology, 2013 vol 23, issue 6, pp279-288 DOI:https://doi.org/10.1016/j.tcb.2013.01.005  COATED VESICLES Coated vesicles self-assemble from simple geometric principles. Not unlike viral capsids! Assembly principles of clathrin, COPII, and COPI coats. Structures of protein cages from clathrin (EMD-5119), COPII (EMD-1232), and a COPI-coated vesicle. The boxes show the vertices of the lattices where the position and orientation of repetitive units are symbolized by arrows. Trends in Cell Biology, 2013 vol 23, issue 6, P279-288 DOI:https://doi.org/10.1016/j.tcb.2013.01.005  CLATHRIN ENDOCYTOSIS The clathrin molecule is composed of a heavy chain (~190 kDa) and a smaller light chain (~25 kDa). Three clathrin heavy and light chains (CHC and CLC) form a trimeric clathrin ‘triskelion’. In a triskelion, the heavy chains interact with each other in the central hub and protrude outwards as long legs. These legs interact with other triskelia to form polygonal lattices, the structural backbone of the clathrin coat. Nature Reviews Molecular Cell Biology, 19(5), 313–326. doi:10.1038/nrm.2017.132  CLATHRIN ENDOCYTOSIS – ADAPTORS CHC – Clathrin Heavy Chain; CLC – Clathrin Light Chain Alpha chain (α) binds to PIP2 Dynamin – a multidomain GTPase that allows “pinching” of the neck of the budding vesicle In the non-PI(4,5)P2-bound conformation the cargo-binding sites on the AP2 are obscured! CHECK THE HIDDEN SLIDES INCLUDED IN THE EXTENDED MOODLE VERSION FOR MORE DETAILS IF INTERESTED! Conner, S. D. & Schmid, S. L. Regulated portals of entry into the cell. Nature 442, 37-44 (2003) doi:10.1038/nature01451.  COATED VESICLES – the GTPase connection Protein Coat assembly drives vesicle formation. Both the COPII (left) and COPI coats are directed in their assembly by small GTPases of the Arf/Sar1 family. Guanine Nucleotide Exchange Factors (GEFs) (Sec12 in COPII or Gea1/2 COPI) activate the small GTPases (Sar1-GTP for COPII or Arf1-GTP for COPI respectively) ANTEROGRADE (Golgi-to-ER) RETROGRADE – ER-to-Golgi Ras family small GTPase  COPs and direction of traffic Sorting into the retrieval (retrograde) pathway is mediated by COPI vesicles, which capture ER residents via their retrieval signals: K(X)KXX for membrane proteins and K/HDEL for lumenal ER residents. COPI COPII animals Yeast and plants COPII COPI COPII COPI COPII vesicles may fuse directly with the Golgi, as occurs in yeast or plants, or may fuse together on their way to the Golgi to generate a pleiomorphic compartment called the ER-Golgi intermediate compartment, or ERGIC (also known as VTC for Vesicular Tubular clusters). Gomez-Navarro & Miller, COP coated vesicles. Current Biology, 2016, 26, R54-57  COATED VESICLES – a closer look at the coats Outer coat Inner coat Sar1 (cargo binding) GTPase (GTP-bound form allows the binding of the cargo by the coat) Gomez-Navarro & Miller, COP coated vesicles. Current Biology, 2016, 26, R54-57 COATED VESICLES – COPII organisation Sec13/31 cage (outer cage) Figure 13-13 Molecular Biology of the Cell (© Garland Science 2008)  COP I cargo recruitment and ArfGAP COPI recruitment to membranes occurs in only two easy steps: binding of activated ADP-ribosylation factor 1 (Arf1) to membranes (via a short amino-terminal amphipathic helix), and the subsequent recruitment of coatomer, which in turn interacts with cargo proteins. Again, soluble lumenal proteins require a cargo receptor — the KDEL receptor — to bridge an interaction with the coat. ArfGAP – (ADP-ribosylation factor GTPase-activating protein) is GTPase-activating protein (GAP) which associates with the Golgi apparatus and interacts with ARF1. It attaches to the budding complex and is stimulated by curvature of membrane. ArfGAP promotes hydrolysis of ARF1-bound GTP and causing the dissociation of coat proteins from Golgi-derived membranes and vesicles. Dissociation of the coat proteins is required for the fusion of these vesicles with target compartments. ArfGAP’s activity is stimulated by phosphoinositides and inhibited by phosphatidylcholine. You can see more details in the hidden bonus slides included in the extended Moodle version! Gomez-Navarro & Miller, COP coated vesicles. Current Biology, 2016, 26, R54-57  Relationships between coats Free online content: Lee & Goldberg; Cell, Volume 142, Issue 1, 9 July 2010, Pages 19-21 Structure of Coatomer Cage Proteins and the Relationship among COPI, COPII, and Clathrin Vesicle Coats. https://doi.org/10.1016/j.cell.2010.05.030  Relationships between coats Common structural modules are found in Sec13–Sec31(COPII) and α–β’-COP (COPI): each subcomplex comprises two β-propeller domains followed by an α-solenoid domain. Interestingly, this characteristic organization can also be found in the clathrin coat and in nuclear pore proteins, reflecting the common evolutionary origin for these proteins, which are all involved in shaping cell membranes. Schematic diagram compares the vertex geometry of COPI, COPII, and clathrin cages. The inner β-propeller domains that form vertex contacts are drawn as orange cylinders, the outer β-propeller domains as yellow cylinders, and the α-solenoid domains as linked green hexagons. Free online content: Lee & Goldberg; Cell, Volume 142, Issue 1, 9 July 2010, Pages 19-21 Structure of Coatomer Cage Proteins and the Relationship among COPI, COPII, and Clathrin Vesicle Coats. https://doi.org/10.1016/j.cell.2010.05.030  Relationships between coats and the nuclear Recent Advances! pore proteins Sec13 is a common component of both Nup and COPII-complexes! NPC – nuclear pore complex NPR – nuclear pore receptors NTRs – nuclear transport receptors Journal of Cell Science (2015) 128, 423–429 doi:10.1242/jcs.083246  VESICULAR FUSION - SNARES Matching the keys or cellular ping-pong? SNAREs = SNAP (Soluble NSF Attachment Protein) REceptor") V-SNAREs = vesicular SNAREs T-SNAREs = target SNARES During membrane fusion, v-SNARE and t-SNARE proteins on separate membranes combine to form a trans-SNARE complex, also known as a "SNAREpin".  VESICULAR FUSION – SNARES - EXAMPLE v-SNARE (synaptobrevin aka VAMP2) and t-SNARES (synataxin 1A and SNAP 25). Synaptotagmin – Ca2+ sensor; triggers the SNARE fusion that leads to the RELEASE of the neurotransmitter. Ca2+ itself is released by the Voltage- activated Ca2+-channels (Cav) upon arrival of the action potential. Synaptotagmin's role in neurotransmitter release likely involves Ca2+-induced conformational transition Zhe Wu, Klaus Schulten. Biophys. J. 107:1156-1166 (2014) A recent review for those interested! https://www.sciencedirect.com/science/article/abs/pii/S0022283620304496  VESICULAR FUSION – NSF NSF (N-ethylmaleimide-sensitive factor) is an homohexameric AAA ATPase. Essential in the synapse! VESICULAR FUSION – TETERING AND RABS Rabs and the cellular membrane postcode Localisation of different Rab GTPases on cell compartments. Over 70 Rabs known in humans! Rab effectors: Cargo budding, selection, and coating Vesicle Transport Vesicle Uncoating and Tethering Vesicle Fusion Harrison S., https://doi.org/10.1016/j.virol.2015.03.043  Rabs and the cellular membrane postcode Rab family of small GTPases control the safe arrival of cargoes at destination ON Rab effectors: Cargo budding, selection, and coating Vesicle Transport Vesicle Uncoating and Tethering Vesicle Fusion OFF The GTP-bound 'active' conformation is recognized by multiple effector proteins and is converted back to the GDP-bound 'inactive' form through hydrolysis of GTP, which is stimulated by a GTPase-activating protein (GAP) and releases an inorganic phosphate (Pi). Newly synthesized Rab, in the GDP-bound form, is recognized by a Rab escort protein (REP). Effector proteins could be – tethers, motors, receptors. Nature Reviews Molecular Cell Biology volume10, pages513–525 (2009)  Rabs and the cellular membrane postcode Rab family of small GTPases control the safe arrival of cargoes at destination Rab effectors: Cargo budding, selection, and coating Vesicle Transport Vesicle Uncoating and Tethering Vesicle Fusion The GTP-bound 'active' conformation is recognized by multiple effector proteins and is converted back to the GDP- bound 'inactive' form through hydrolysis of GTP, by a GTPase-activating protein (GAP) The geranylgeranylated, GDP-bound Rab is recognized by Rab GDP dissociation inhibitor (GDI), which regulates the membrane cycle of the Rab (extracting it from the membrane). Targeting of the Rab–GDI complex to specific membranes is mediated by interaction with a membrane-bound GDI displacement factor (GDF). Effector proteins could be – tethers, motors, receptors. Vos et al., 2019 https://doi.org/10.1021/acs.biochem.8b00932  Rabs and the cellular membrane postcode Rabs create microdomains within compartments and can be used as markers for cellular targeting The occurrence of microdomains enriched for specific Rab GTPases, as exemplified with endosomes. Nature Reviews Molecular Cell Biology volume10, pages513–525 (2009)  VESICULAR TRAFFIC & FUSION SNAREs, GTPase switch mechanism COPII, Sec 61 SNAREs, LDL receptor in coated vesicles  Autophagy Targetting membranes for (self)-degradation Stress signal = Atg6 Atg8 = To date some 40 Apg/Atg proteins have been discovered Autophagy is important for cell-recycling, phagocytosis and apoptotic cell death American Journal of Physiology - Lung Cellular and Molecular Physiology Published 15 July 2013 Vol. 305 no. 2, L93-L107 DOI: 10.1152/ajplung.00072.2013  Autophagy After the ubiquitin-like protein Atg8 (GABARAP/LC3/GATE16) has had its C-terminal arginine residue cleaved by the cysteine protease Atg4, it is passed on to E1 (Atg7) and E2 (Atg3) and transferred into the head group of its substrate phosphatidylethanolamine (PE). This Atg8-PE conjugate functions as part of the membrane component of the autophagosome. When Atg8-PE is once again deconjugated PE by Atg4, the Atg8 is recycled. Incidentally, an E3-like protein in autophagy has not yet been found. Bavro et al. EMBO Rep. 2002  Autophagy Nobel Prize 2016 – Yoshinori Ohsumi  ESCRT systems Endosomal Sorting Complex Required for Transport (ESCRT) machinery is an evolutionarily-conserved, multi-subunit membrane remodeling complex Vsp4 (vacuolar protein sorting 4 AAA–ATPase) - similar to NSF Simplified mode of action of ESCRT and ESCRT-related components during endosomal vesiculation and creation of Multivesicular Bodies (MVBs). ESCRT proteins help create internal vesicles in MVBs (multi vesicular bodies) and then disconnect from the vesicle when finished. The Vps4/SKD1 complex is an AAA ATPase, disassembles the complex and plays a role in final stages of the budding process. Wollert, T. & Hurley, J. H. Molecular mechanism of multivesicular body biogenesis by ESCRT complexes. Nature 464, 864-869 (2010).  ESCRT systems Endosomal Sorting Complex Required for Transport (ESCRT) machinery is an evolutionarily-conserved, multi-subunit membrane remodeling complex Originally identified in yeast for its essential role in the biogenesis of intraluminal vesicles (ILVs) upon a class of endosome called the multivesicular body (MVB) – or late endosome. ESCRTs recognise the ubiqutin-tagged transmembrane proteins which are marked for degradation. Thus, are important for the lysosome formation. ESCRT-III is the hypothesised membrane fission complex within the ESCRT-machinery and is directed to its sites of action through interaction with upstream ESCRT-components. ESCRT-III is involved in nuclear envelope reformation, cytokinesis and in cell division. Receptor internalisation – e.g. LDL (principal cholesterol transport from blood to cells); EGFRs (epithelial growth factor receptors) and similar. Current Opinion in Cell Biology 2016, 38:1–11 Free full text available online: http://dx.doi.org/10.1016/j.ceb.2015.12.001  ESCRT systems ESCRT system often gets hijacked by budding viruses – e.g. HIV-1 ESCRT-III filaments follow similar structural principles. A. CHMP2A spirals (negative stain); B. CHMP2B dome surrounded by membrane (negative stain); HIV-1 buds from the plasma membrane and recruits ESCRT proteins including ESCRT- C. CHMP2A-CHMP3 dome (cryo-EM); III and VPS4. ESCRT-III CHMP2A-CHMP3 form helical polymers in vitro (lower left: cryo D. CHMP2B membranous bottleneck (cryo-EM). EM structure at 22 Å resolution) that might assemble inside the neck of the bud and coil Scale bars are 50 nm. up to form dome-like structures that lead to neck constriction that will favor membrane fission. Additional energy for the final budding process may be provided by the ATPase VPS4. Middle panel: model of ESCRT-III induced membrane neck constriction; Gag inside the virion; ESCRT-III polymers in the neck: green CHMP4 filaments coordinated by dimeric Alix (V-shaped) and red CHMP2A-CHMP3 filaments). Effantin, G., Dordor, A., Sandrin, V., Martinelli, N., Sundquist, W.I., Schoehn, G., and Weissenhorn, W. (2013). ESCRT-III CHMP2A and CHMP3 form variable helical polymers in vitro and act synergistically during HIV-1 budding. Cell Microbiol 15, 213-226 Thank you for your attention! See you back in Week 7!  Evolutionary connections Here we describe the ‘Asgard’ superphylum, a group of uncultivated archaea that, as well as Lokiarchaeota, includes Thor-, Odin- and Heimdallarchaeota. Asgard archaea affiliate with eukaryotes in phylogenomic analyses, and their genomes are enriched for proteins formerly considered specific to eukaryotes. In addition, thorarchaeal genomes encode Notably, thorarchaeal genomes encode several homologues of eukaryotic homologues of eukaryotic Sec23/24 family proteins, membrane-trafficking machinery components, including Sec23/24 and TRAPP which are essential components of COPII, a protein domains. complex responsible for vesicle-mediated ER-to-Golgi transport of protein cargo. So, what defines the Eukaryota!? Not the actin; not the tubulin cytoskeleton; nor the profilin or the endomembrane/COP systems! Lokiarchaeum, an archaeon whose genome encodes small GTPases related to those used by eukaryotes to regulate membrane traffic. 100+ putative small Ras-like superfamily GTPases, six actin genes, a strikingly eukaryote-like ribosome, and clearly detectable ESCRTIII, ESCRTI, and ESCRT0 complexes. While clathrin itself hasn’t been found yet, there is a dynamin precursor in the genome. Archaea such as Ignicoccus hospitalis, along with several types of bacterium, have independently evolved endomembrane systems!  Evolutionary connections COP/Sec/SNAREs/small GTPases Nature 3 Oct 2018 https://doi.org/10.1038/d41586-018-06868-2 Approx 1.8 Billion years ago The Eukarya (organisms whose cells harbour DNA in a nucleus) are thought to have arisen from a merger between their last archaeal ancestor and a bacterium. In addition to a nucleus, eukaryotes have several characteristics that are thought to separate them from archaea, including: a complex internal system of membranes called endomembranes; the actin cytoskeleton, the dynamics of which are regulated by the protein profilin; and energy-producing organelles called mitochondria, which arose from the symbiotic bacterial partner. But Akıl and Robinson provide evidence that members of the Asgard superphylum — an extant group of archaea thought to be related to eukaryotes — harbour a primitive profilin-regulated actin cytoskeleton.  Evolutionary connections https://knowablemagazine.org/article/living-world/2023/how-endomembrane- system-of-eukaryotic-cells- evolved?fbclid=IwAR2Mgnz61aU42hedPoh4aS7p20dyjKg-uhEde1oABJoGl9a- xXYaVxmW3CM