Nakayama, K.I. and Nakayama, K. (2006) Ubiquitin ligases: cell-cycle control and cancer. Nat Rev Cancer, 6,

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1 Molecular Machines Lecture 4: Protein degradation, membrane insertion and export. Autumn 2013, Michael Pavlov Based on Chapters 13.(1-3) and 14.(1-4), MCB book Protein Ubiquitinylation Protein Degradation by Proteosome tmrna Making proteins for export and membrane insertion Delivery of the translating ribosome to the translocon Ribosomal tunnel and the translocon Translocon and N-linked (core) glycosylation Folding of proteins after glycosylation Unfolded protein response (UPR) General principles of vesicle mediated trafficking Addressing transport vesicles in the cell

2 Protein Ubiquitinylation and Degradation E1 Ub-activating enzyme activates a small 76 AAs (8 kda) protein ubiquitin (Ub) by first attaching AMP to the C-terminus of Ub (containing Gly), which results in a protein-adenylate. Then, E1 transfers Ub from AMP to E1 Cys side chain forming a thio-ester with Ub. There are two E1 enzymes in human cells and one of them (Ube1) is essential. E1-Ub exposes its binding site for one of the 60 E2 conjugating enzymes present in the human cell. Ub is then transferred from Cys of E1 to an active Cys of E2 in a trans-esterification reaction. From E2, Ub is transferred to a Lys of a target protein in the reaction catalyzed by specific E3 ubiquitin- protein ligases. SCF and APC ligases are important for cell cycle progression Nakayama, K.I. and Nakayama, K. (2006) Ubiquitin ligases: cell-cycle control and cancer. Nat Rev Cancer, 6,

3 E3 Ub-ligases Two major types of E3 Ub-ligases. The RING-type E3 Ub-ligases transfer Ub from E2-Ub conjugating enzyme directly to a substrate protein. There are more than 500 different RING-type E3-s in humans specific for different protein substrates. The HECT type E3-s have an active Cys which is first thio-esterified with Ub and then E3 transfers Ub to its cognate substrate protein. The number of different HECT is much smaller, about 50. E3-ligases can run both mono-and poly-ubiquitinylation of their substrates. E3 works processively holding the substrate protein and adding Ub sequentially to a Lys side chain of the previously added Ub. Li, W. and Ye, Y. (2008) Polyubiquitin chains: functions, structures, and mechanisms. Cell Mol Life Sci, 65, Lys chain of the Ub already attached to a protein substrate (or to a Lys chain of an original protein substrate) attaches the next Ub by the amino-attack (from K48 or K63) on the thio-ester of the Ub attached to E2 or E3. The chemistry of this reaction is quite similar to peptidyl-transfer reaction run by the ribosome. Mono-Ub serves mainly regulatory purposes and does not target proteins for degradation. Poly-Ub >4 is requires for the ubiquitinylated protein to interact with the 26S proteosome.

4 The proteosome The proteosome: 20S core The proteosome is a large molecular machine of about 2.5 MD composed of about 33 different types of proteins. It has a size comparable with that of the ribosome. The catalytic 20S core is enlarged by the attachment of regulatory 19S cap(s) (PA700) in the 26S proteosome. The 20S catalytic subunit contain two inner rings each containing 7 slightly different subunits with 3 proteolytic active sites per ring, facing the inner chamber. Those cleave the protein after acidic ( 1), basic ( 2) or hydrophobic ( 5), residues. The outer -rings (which also contain 7 different subunits) gate the substrate protein into the inner chamber.

5 The core 20S proteosome Stadtmueller, B.M. and Hill, C.P. Proteasome activators. Mol Cell, 41, 8-19 In some cells (immune-system) i-type of 1,2 and 5 is produced. Immune-proteosome has enhanced cleavage after hydrophobic residues and reduced after acidic ones: this is important for antigen presentation; 5t is specific for thymus cells. In eukaryotes -subunits (and -subunits) are all different. In archeae and some bacteria only one type of and one type of -subunits are made The N-terminus of -subunits blocks the entry into the 20S by forming a closed gate.

6 ATP-dependent Proteosome Striebel, F., Kress, W. and Weber-Ban, E. (2009) Controlled destruction: AAA+ ATPases in protein degradation from bacteria to eukaryotes. Curr Opin Struct Biol, 19, S cap of 26S proteosome consists of at least 19 protein subunits 6 of those are ATPases that mainly carry out protein unfolding. Other subunits bind to substrate (Rpn10/13) DUBs (De-UBiquitinilases), like the Rpn11 remove Ub- from the protein allowing the Rpt ring of the 19S particle to unfold the target protein and thread the unfolded polypeptide chain into the destruction chamber of the 20S. ClpA-ClpP and HslU-HslV are bacterial analogous of 26S proteosome. Actinomycetaes use unstructured Pup (Prokaryotic Ub like Protein) as a degradation tag (attached through C-term Glu side chain to a Lys of a target).

7 Ub receptor subunits of Rpn 10 and Rnp 13 recognizes poly-ub proteins either directly, or through adaptor proteins which contain Ub-like domains. Rpn11 removes Ub-marks from the protein allowing the 19S particle to unfold the target protein. ClpS recognizes YLA-containing peptides destined for fast turn-over in bacteria. Striebel, F., Kress, W. and Weber-Ban, E. (2009) Controlled destruction: AAA+ ATPases in protein degradation from bacteria to eukaryotes. Curr Opin Struct Biol, 19,

8 ATP-dependent unfolding AAA+ hexameric rings unfold proteins using ATP energy. Unfolding involves ATP fueled movements of GYVG loops Protein substrate is unfolded and pooled inside the destruction chamber of Clp, HlsV or 20S. The model suggests that the YV hydrophobic motive grips the peptide patch and pulls it down. The pulling force leads to the unfolding and translocation. Striebel, F., Kress, W. and Weber-Ban, E. (2009) Controlled destruction: AAA+ ATPases in protein degradation from bacteria to eukaryotes. Curr Opin Struct Biol, 19, Subunit cycling drives translocation In this model the opposite loops from para-subunits work in concert. Subunits of the AAA+ hexa -ring can acquire conformations with high, low and no ATP/ADP affinity. Upon ATP hydrolysis the two loops move down, puling the peptide, the ADP bound subunit looses ADP/ATP affinity and former ATP/ADP free subunit binds ATP. Then the cycle repeats.

9 Rescue of stalled ribosomes by tmrna Moore, S.D. and Sauer, R.T. (2007) The tmrna system for translational surveillance and ribosome rescue. Annu Rev Biochem, 76,

10 ClpXP proteosome in bacteria ClpP compartment Sauer, R.T., et al. (2004) Sculpting the proteome with AAA(+) proteases and disassembly machines. Cell, 119, 9-18 The tag sequence is recognized by SspB adaptor protein that targets it to ClpXP degradation machines in E. coli that destroy the tag together with truncated protein. ClpXP also recognizes the ALAA C-terminus of the tag directly. ClpXP is similar to ClpAP. ClpAP can degrade tagged proteins as well.

11 Making proteins for export and membrane insertion Cell surface receptors, ion channels and other proteins that start their synthesis in the cytoplasm but end up as inner membrane proteins on the cell surface or become exported from the cell. About 40% -50% proteins in high organisms are either membrane proteins or exported from the cell. Their journey to the cell surface proceeds in the following steps: selection by the Signal Recognition Particle (SRP) delivery to the translocon export or membrane insertion passage through the Golgi compartment to the cell surface The final destination of proteins is determined by the signals that are coded in their own aminoacid sequences. Those signal sequences are recognized by a variety of different receptors in the cell, by SRP in particular. The signal sequence (SS) is recognized by SRP. The ribosome in the complex with SRP attaches to the SRP receptor associated transiently with the translocon. Upon binding of the ribosome:srp complex to the receptor, GTP is hydrolyzed both on SRP and the receptir SS is transferred from the SRP to the SS recognition site on Sec61a translocon subunit). After the recognition of the SS by the translocon the ribosome binds to it firmly and the translation resumes

12 Signal sequences for the delivery to the translocon by SRP have a hydrophobic core flanked by basic aminoacids at the N end, or at both the N- and C- ends in the case of trans-membrane segments. The length of the hydrophobic core is typically between 8 and 15 AAs. In the cytoplasm, upon emerging from the exit of the ribosomal tunnel at the back of the 60S subunit of the ribosome, the nascent peptide interacts first with the Nascent (Chain) Associating Complex (NAC) that binds to all nascent peptides (eukaryotic NAC corresponds to the trigger factor (TF) in prokaryotes) and with SRP.

13 Structure of SRP SRP54 protein in eukaryotes and ffh protein in prokaryotes bind domain IV of SRP RNA and forms the core of the SRP particle that interacts directly with the signal sequence. Mammalian SRP consists of one 7S RNA and 6 different proteins. In eukaryotes SRP has additional elements (Alu) with an additional function: SRP binding to the ribosome arrests protein synthesis until the docking to the translocon occurs. The arrest is due to the block of the site on the ribosome to which elongation factor EF1 and EF2 bind by SRP9/SRP15 complex at the Alu end of SRP. Egea, P.F., Stroud, R.M. and Walter, P. (2005) Targeting proteins to membranes: structure of the signal recognition particle. Curr Opin Struct Biol, 15,

14 Ribosomal tunnel and the translocon The peptide bonds are made in the peptidyl-transferase (PT) center situated at the bottom of a large crevice in the 60S subunit. The peptides goes through the tunnel (about 7 nm long) through the 60S subunit and exits on the back of the 60S as shown in the cross-section through the 60S subunit with gray ribosomal RNA and green ribosomal proteins. The 40S (not shown) is to the left from the 60S so that it faces the PT center. The Translocon is well conserved from bacteria to humans. Cross, B.C., Sinning, I., Luirink, J. and High, S. (2009) Delivering proteins for export from the cytosol. Nat Rev Mol Cell Biol, 10, The basic translocon consists of SecY, SecE and Sec in archaea which are homologues to mammalian: Sec61, Sec61 and Sec61. It can open laterally.

15 The co-translational translocon that works with the ribosome contains the Translocon Associated Protein TRAM. It also contains: One copy of Signal Peptidase (SP) that cleaves off the signal peptide One copy of Oligo-Saccharyl-Transferase (OST) that glycosylates the nascent peptide at Asn in the tri-peptide sequence Asn-X-Ser/Thr. Resting translocon must maintain the permeability barrier between the ER lumen and the cytoplasm. This is the function of the short helical plug shown in the figure. After the binding of the ribosome and the resumption of the translation the nascent peptide eventually enters the lumen. This means that the translocon pore should open. The opening of the translocon occurs when the nascent peptide reaches AAs in length. This opening is accompanied by a major conformational change in the translocon with the plug moving away to the lumen of the ER, opening the tunnel. Rapoport, T.A. (2007) Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. Nature, 450,

16 The ribosome forms a tight seal with the opened translocon so that the permeability barrier is maintained. Small molecules from the ER lumen may travel as far as the peptidyl-transferase center of the ribosome (but not further), which means that the ribosomal peptide conducting tunnel and the translocon pore form a continuous tunnel for the nascent peptide. Open translocon. Here the plug is removed and the nascent peptide can be pushed into the ER-lumen upon the resumption of translation. The ring of the Ile residues blocks the movement of small molecules between the ER and the cytoplasm when the nascent peptide moves through the opened pore.

17 Post-translation protein translocation into the ER. In contrast to high eukaryotes in which the ER delivery is predominantly co-translational, many yeast proteins are delivered to the ER after being completely synthesized on the cytoplasmic ribosomes. Post-translational ER translocation occurs on specialized translocons that are modified by the presence of the tetrameric Sec62/63 complex BiP-ATP binds to J-domain of Sec63. This binding promotes a conformational change resulting in ATP hydrolysis and BiP-ADP binding to the luminal portion of the peptide to be translocated. The peptide with bound BiP-ADP is caught in the ER lumen and can not slide back into the cytoplasm. Eventually it will slide forward so that the next BiP-ADP can be loaded by Sec63. Sequential BiP-ADP loading onto the peptide would effectively pool it into the lumen of the ER and would simultaneously result in its folding by BiP inside the lumen. BiP-ADP would eventually exchange ADP for ATP and dissociates from the peptide. BiP-ADP acts as a Brownian ratchet in pooling the protein inside the lumen.

18 Protein insertion into the membrane. Membrane proteins can be inserted in many different ways The insertion principle are best understood for the single pass proteins with only one transmembrane fragment. Those can be sub-divided into three types depending on whether the N or the C-terminus is the cytosole and on whether the bulk of the protein is in the cytosole on not. The protein translocation proceeds until a stop transfer sequence (usually a hydrophobic helix containing about 22 hydrophobic amino-acids) is synthesized and enters the translocon. It causes the translocon to open laterally, allowing the helix to enter the lipid bi-layer. After this, the ribosome may disengage from the translocon and continue the synthesis of a rather short C-terminus in the cytoplasm. Alternatively the ribosome may remain on the closed translocon while the synthesized peptide loops out into the cytoplasm. The protein synthesis terminates then in a usual way.

19 Translocon and N-linked (core) glycosylation. The cell produces the core glycan consisting of 14 saccharides in a complex sequence of consecutive reactions that are catalyzed by enzymes situated on the both sides of ER membrane (cytoplasmic and lumen sides). Fig Hemagglutinin folding and assembly. The attachment of the glycan to the nascent protein on the lumen side of the ER is carried out by OST (Oligo- Saccharyl-Transferase). OST glycosylates the nascent peptide at (almost) every Asn in the tri-peptide sequence Asn-X-Ser/Thr after the nascent peptide protrudes by AAs from the exit of the translocon. OST is situated laterally to the translocon and contact the ribosome bound to translocon.

20 Helenius, A. and Aebi, M. (2004) Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem, 73, Several cycles of glucosylation: de-glucosylation may be required until a glycoprotein folds properly. ERGIC-53 is a membrane bound receptor that binds to a properly folded de-glycosilated glycoprotein with Glc0Man9GlcNAc2 as well as with Glc0Man8GlcNAc2 form of the core glycan (with mannose removed by mannosidase I). Removal of the middle mannose that occurs after several folding trials also makes the protein susceptible to a quality control induced degradation. Calnexin/Calreticulin Folding cycle of glycoproteins Glycan is first de-glucosylated by Glucosidases I and II. Glycoproteins with mono-glucose-glycans can bind to CNX and CRT that have high affinity to this form of the glycan. Misfolded proteins bind near the hydrophilic finger (P-domain) of calnexin). Here the hydrophobic patches of the misfolded peptide may be forced into their proper conformations. PDI homolog ERp57 forms a complex with CNX and CRT and can reshuffle the SS bonds in bound glycoproteins. Upon spontaneous release from CNX or CRT the glycan is de-glucosylated completely by Glucosidase II. UDP-glucose:glycoprotein Glucosyl Transferase (UG-GT) recognizes deglucosylated glycans in partially folded proteins and attaches glucose back to those glycans. They rebind then to CNX/CRT Thus UGGT is one of folding sensors in the lumen that also promotes the glycoprotein folding.

21 Unfolded protein response (UPR) Changes in the lumen environment, like the presence of excess of unfolded or improperly folded proteins are first sensed by the BiP chaperon. Three known secondary sensors participate in the response at the transcriptional and translational levels. UPR at the transcriptional level (I) Upon UP (unfolded protein) stress, BiP binds unfolded proteins and dissociates from Ire1a. This leads to Ire1a dimerisation after which the Ire1a autoactivation occurs through the cross-phosphorylation of the Ire1a dimer on he cytoplasmic side. The phosphorylation of Ire1a activates its C-terminal T- domain which has a sequence specific L-like RNAse activity directed against XBP1 mrna. XBP1 mrna resides in the cytoplasm. The T domain of Ire1a excises a 26 nucleotides intron from the middle of XBP1 mrna. Zhang, K. and Kaufman, R.J. (2006) The unfolded protein response: a stress signaling pathway critical for health and disease. Neurology, 66, S The spliced mrna codes for an XBP1 transcription factor. The intron excision results in a frame shift and in the production of an altered, extended C-end of the XBP1 transcription factor. The mature XBP1 translocates into the nucleus and binds to the UPR response element for XBP1 in the DNA CCAAT(N)9CCACG and activates chaperon genes like BiP and calreticuline.

22 Zhang, K. and Kaufman, R.J. (2006) The unfolded protein response: a stress signaling pathway critical for health and disease. Neurology, 66, S UPR at the transcriptional level (II): ATF6 dependent activation of UPR The ATF6 regulation of the UPR pathway exists only in high eukaryotes and works in parallel with the Ire1a/XBP1 pathway. In response to the BiP dissociation from the ERluminal domain of ATF6, Golgi-localization signals (GLS1 and GLS2) on the luminal domains of ATF6 open and ATF6 is transported to the Golgi where it is cleaved by S1P/S2P proteases so that its cytoplasmic domain (p50) is released from the Golgi membrane into the cytoplasm. Its NLS opens and ATF6 p50 is imported into the nucleus by one of the Importins (Ran-dependently). The p50 domain of ATF6 is a b-zip transcription factor which binds to the same response element as XBP1 and activates a similar set of ER-chaperons.

23 UPR at translation level: PERK kinase Another protein involved in UPR is PERK (PRK-like ER-kinase) that spans the ER membrane between the ER lumen and the cytoplasm. The luminal domain of PERK is similar to that of Ire1: Schroder, M. and Kaufman, R.J. (2005) The mammalian unfolded protein response. Annu Rev Biochem, 74, Zhang, K. and Kaufman, R.J. (2006) The unfolded protein response: a stress signaling pathway critical for health and disease. Neurology, 66, S While the general level of translation is greatly repressed, the translation of several mrnas including the mrna of transcription factor ATF4 is selectively increased by a mechanism not well understood. The induction of ATF4 leads to the expression of different proteins including those participating in apoptosis, protein trafficking and oxidation. If the unfolded protein persists the cell commits apoptosis. When bound to BiP the kinase is monomeric but it dimerases in the ER lumen upon BiP dissociation. Upon dimerisation PERK is activated by cross phosphorylation that occurs on the cytoplasmic side Activated PERK phosphorylates eif2. Phosphorylation of Ser 51 in the -subunit of IF2 drastically reduces the initiation of protein synthesis and general translation. In this way the UP stress in the ER sends a signal to slow down the translation to reduce the protein load in the ER.

24 Three major types of coated vesicles. It is currently believed that Golgi cisternae physically move in the direction from cis- to trans- Golgi acquiring specific enzymes by retrograde vesicle traffic driven by COPI. One major rout of the vesicle transport that carries export-destined proteins runs between the ER-lumen and Golgi. Note that Cis-Golgi network=ergic. This transport is carried out by COPII vesicles. The assembly, targeting and fusion to the target membranes are very similar for different vesicles with the COPII vesicles being best understood. The transport of a protein from ER-to cis-golgi takes min, while the time to reach the cell surface is about 60 min. The protein spends in Golgi at least 15 min.

25 Formation of COPII vesicles Figure The start of COPII vesicle assembly In response to the presence of cargo proteins destined to the Golgi a transmembrane Sec12 factor exchanges GDP for GTP on Sar1 protein. This changes Sar1 conformation making Sar1 to expose its N-terminal hydrophobic anchor through which it binds to the cytosolic face of the ER membrane. Sar1 on the membrane forms a binding site for the Sec23/Sec24 coating protein complex Sec23/Sec24 also recognizes particular sequences in the cytoplasmic domains of activated cargo receptors or membrane cargo proteins (like di-acidic Asp-X-Glu (DXE) sequence or its extended variant: YxDxE ): Sec13/ Sec31 coatomers also bind at this stage and stabilize the inner Sec23/24 coat. Coating proteins recognize integral membrane v-snare proteins and include them into the vesicle. After the completion of the. assembly the vesicle buds out of the ER membrane

26 The A-site of Sec24 recognizes YNNSNPF sequence while the B-site recognizes the DXE motive of the cargo The C-site recognizes the LXXLE motives present in a Bet v-snare protein. Several different iso-forms of Sec24 are present in high eukaryotes with presumably different binding sites. This may explain a large variety of export signals for the COPII inclusion. Mancias, J.D. and Goldberg, J. (2005) Exiting the endoplasmic reticulum. Traffic, 6, After the budding the GTP hydrolysis occurs on Sar1 and Sar1 looses its affinity to the coating proteins. The coat disassembles exposing v- SNARE to recognize t-snare proteins in the target membrane.

27 Addressing transport vesicles in the cell The receiving membrane has a complex of membrane proteins (t-snare-s) that serves as a receptor for a particular v-snare. Specific interactions between the t-and v- SNARE proteins provides the major contribution into the specificity of vesicle targeting. In addition to v-snare, vesicles carry also Rab proteins that bind to the vesicle surface after the vesicle sheds its coat. Upon vesicle delivery its Rab*GTP binds to a specific Rab receptor on the surface of a target membrane, which increases the addressing specificity. A tight docking occurs due to the formation of 4-helix bundle when one v-snare (VAMP here) interacts with t-snare (SNAP 25 here) plus Syntaxin (here) as shown in the figure. In other cases, v-snare can interact with three monomeric t-snare proteins in the receiving membrane. The t-/v- SNARE bundle is parallel. It formation results therefore in membrane proximity. After the fusion the trans-snare complex (with t- and v- helix coming from different membranes) becomes a cis-complex without the helix bundle unwinding.

28 Kirchhausen, T. (2000) Three ways to make a vesicle. Nat Rev Mol Cell Biol, 1, Assembly of COP I vesicles The assembly of COPI vesicles is similar to that of COP II. First, a GTP/GDP exchange factor interacts with an ARF protein which is an analogue of the Sar1. ARF*GDP is weakly bound to the Golgi membrane through its myristoyl anchor. ARF*GTP changes its conformation and exposes its N-terminal hydrophobic segment, which anchors ARF*GTP firmly to the cis-golgi membrane. This ARG*GTP binding initializes the assembly of COPI coatomers complexes containing 7 protein subunits (total MW around 340 kd). The receptors and membrane proteins that have affinity to COPI coatomers (those, for example, that have a di-basic signal sequence KKXX, like in ERGIC53 receptor) are selected through interactions with COPI a and b subunits. The COPI coated vesicle buds out and then GTP is hydrolyzed to GDP on the ARF protein. Then the COPI coat proteins de-polymerase and the vesicle becomes uncoated exposing its v- SNARE proteins. The un-coating is necessary for the fusion with the target membrane.

29 KDEL receptors and COP I coated vesicles: COPI directed retrograde transport to maintain the composition of the ER lumen. Proteins that belong to the ER lumen often have KDEL (Lys-Asp-Glu-Leu) sequence at their C-terminus. When those are occasionally picked up by the COPII vesicles and delivered into the cis-golgi they are selectively retrieved and transported back to the ER lumen. KDEL receptors in the cis-golgi membrane bind proteins with the C-terminal KDEL sequence. Those receptors are recognized by a COPI coating protein complex and included into the COP I during their budding from the cis-golgi. COPI vesicles pinch out from the cis- Golgi, shed their coats and move back to the ER, fuse with the ERmembrane and release proteins with KDEL signal sequences into the ER lumen. The binding of the C-terminal KDEL sequence to the KDEL receptor is favored in the Golgi. The binding is very ph sensitive and a small difference in the ph between the ER and the cis-golgi favors the release of KDEL proteins from KDEL receptors in the ER and their strong binding in the cis-golgi. This way the depletion of the ER-lumen of essential lumen proteins through their inclusion (intentional or not) into the COP II vesicles is counteracted.

30 Getting cargo from cell surface Formation and budding of clathrin coated vesicles. Upon cargo recognition, the ARF protein initiates the assembly of the AP coat after which clathrin polymerizes around the AP-type clathrin vesicles. The clathrin coated pit then pinches off in the process that involves G- dependent protein dynamin that polymerases around the vesicle neck. Clathrin cage