CH10_HO - ENDOPLASMIC RETICULUM An organelle adjacent to...

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Unformatted text preview: ENDOPLASMIC RETICULUM An organelle adjacent to the nucleus that consists of sacs (cisternae) and tubules The ROUGH ER, physically associated with ribosomes on its cytosolic surface, is involved in PROTEIN PROCESSING The SMOOTH ER is involved in LIPID SYNTHESIS and METABOLISM 1. mRNA is translated by ribosomes that may be associated with the rough ER 2. The nascent protein is initially driven into the ER (for folding. processing. and molecular associations) 3. Many proteins then migrate within vesicles to the Golgi apparatus (for further processing and O-glycosylation. if applicable) 4. Some proteins are finally sorted for transport to lysosomes, the plasma membrane, or tor secretion by the cell. fiGNALSEQUENCES cleavage N l @m®®9®m9®®®®®®®®®®®®®®®®9®®®m30 TM ~20 amino acids. Signal sequence with typical hydrophobic stretch 10.6 Some examples of signal peptides (continuous hydrophobic sequences are in bold): Preproalhumin l-IKUUTFLLLLEISGSAES Pre-IgGlightchain MDHRAPAQIEGFLLLLEPGTRC Prelysozy’me MRSLLILVLCFLPLAALG Preprolaecin MNSQVSARKAGTLLLLHHSNL Prepenicillinase MSIQHFRVALIPFFAFCLPVFA Prevesicula: scomacicis MKCLLYLAFLF I HVNC virus glycoprocein ?relipoprotein HKATKLVLGAVILGSTLLAG Addition of a signal peptide to the N-terminal portion of cytoplasmic proteins that naturally do not have one causes them to translocate to the ER, where the added signal sequence will be cleaved off. STEPS OF COTRANSLATIONAL TRANSLOCATION TARGETING OF SECRETORY PROTEINS SRP binds the signal sequence. Translation SRP and SRP-R are tRNA . pauses SRP_ribosome displaced and recycled 'l‘ r ‘ complex ’ binds SRP-R /. —¢ on the ER // Translation ‘.-' 1 f ‘ resumes RNA - a 7"- . membrane .. H CO.” Starts ll‘l Signal sequence on nascent protein SRP I CYTOSOL ER LUMEN H SRP-R on the Sec61 channel ER membrane 1. As the polypeptide emerges from the ribosome it binds to a si nal reco nition particle. The SRP binds to both. the polypeptide and the ribosome. Translation stops. 2. SRP binds to the SRP receptor on the ER surface. 3. The polypeptide begins to be driven through the transmembrane Sec61 translocation complex and SRP is released. 4. Translation resumes. The signal sequence is cleaved on the lumenal side of the ER. The rest of the polypeptide translocates to the ER and is released. THE SIGNAL RECOGNITION PARTICLE AND THE SRP RECEPTOR SRP Methionine resudues on F54 that bind to Large P18 signal sequerl‘ces domain cmculscm m,cH,scH. SRP receptor on the ‘cmcmscn, \cmcmsm ER membrane P681??? SR“ SRB Required to! nroleln translocalion NH 2 COOH P9lP14 Small w domain \lnteract With ribosomes AI; 5 NH2 The mammalian signal recognition particle (SRP) consists of at least 6 proteins associated with a 75 RNA, arranged as a rod-like complex. Its small domain is responsible for translational control (inhibition). Its large domain is involved in signal sequence recognition, protein positioning for translocation, and protein binding to the cytosolic face of the ER membrane. One start transfer sequence and one stop transfer sequence I‘l remaindernf " pulgpeplldethaw signal sequence One cleavable signal sequence wgnal J Iranslecelian channel DEDUOBE CflDH SIDD’ \ransrer, sequence EUDH starlr (ransler SEfluEnce l \- e fii’Tc’Jfr—L' w I CVTO 50L u. [R LUHEN The ER translocation channel Whether the cleavable signal sequence remains attached to the ER channel or is released into the phospholipid bilayer prior to degradation remains unresolved. Regardless. the signal sequence will be cleaved by the signal peptidaSe complex. If there is no cleavable signal sequence the first non-cleavable signal sequence will be the “start transfer sequence." It is believed that when a stop-transfer sequence contacts the ER channel. the nascent protein can move laterally into the phospholipid bilayer. Transmembrane proteins with one or more internal, non-cleavable start transfer sequences and stop transfer sequences will have multiple transmembrane segments. Molecular Biology ofthe Cell, 4th ed, Albens et al. SOME EXAMPLES OF TOPOGENESIS cytoplasm mRNA ER lumen ER resident protein N Multi-pass 2TM I I N? Signal peptidase : I Cleavable signal sequence Stop-transfer sequence, (internal, non-cleavable) Start-transfer sequence, (internal, non-cleavable) THE POSITIVE-INSIDE RU LE (where “inside” refers to the cytoplasm) Amino acid residues near the cytosolic side of the hydrophobic TM sequence tend to be more positive than those near the ER lumenal side. Observations: Transmembrane helices are 20 to 25 amino acids long and have a high overall hydrophobic content. This predicts TM domains based on hydrophobicity plots. The frequency of positively-charged Arg and Lys residues is higher in the non- transmembrane segments (loops) on the inner part of the cell. Cytoplasmic side loops contain more positively charged residues than extracellular loops. This predicts orientation. A+: the difference between the number of Arg+Lys on the two sides of the TM helix. The sequence with the largest A+ tends to go inside (i.e., to face the cytoplasm). Hydrophobicity plot "\ "‘\~ . helix , / helix?‘? \ \ I I I I Transmcmbrnnctopology RETRIEVAL PATHWAY TO THE ER Resident ER membrane proteins contain signals that bind to COPI coats and are thus returned. Their C-terminus is Lys Lys Xxx Xxx (KKXX). Soluble ER resident proteins that have left the ER have a different retrieval signal: Lys-Asp-Glu-Leu (KDEL). Evidence? lfthe KDEL sequence is trans erre to a proteint at is normally secreted. this protein is retained in the ER. Conversely, if KDEL is removed from BiP. BiP is secreted. Soluble ER proteins bearing the KDEL retrieval sequence interact with the KDEL-receptor molecule, a transmembrane protein that binds and packages any protein with the KDEL (C-terminal sequence into COPI-coated retrograde transport vesicles. ER-Iesitlem ploiein memhl mle KDEL receptor Lumen Cylosol Ad .1 pm COPI: coat protein | com The KDEL-receptor molecule - The KDEL receptor molecule shuttles between the ER and the Golgi. - To return to the ER, the KDEL-receptor binds KDEL-bearing molecules in the vesicular tubular clusters (or ERGIC) with high affinity, effectively capturing the “escaped” resident ER proteins present at a low concentration. ° Conversely, the KDEL-receptor molecule binds KDEL-bearing molecules with lower affinity in the ER. This is necessary so it can unload cargo picked up at the VTCs and Golgi in spite of the high concentration of KDEL-bearing proteins in the ER. - How can the binding affinity of the KDEL-receptor be different in the Golgi and ER? Because of differences in pH, controlled by H+ pumps. The KDEL-receptor molecule binds KDEL-bearing ligands under slightly acidic conditions in the VTC and Golgi, but releases them at the neutral pH of the ER. pH-SENSITIVE PROTEIN-PROTEIN INTERACTIONS PLAY KEY ROLES IN MANY OF THE SORTING STEPS IN A CELL Retrieval of KDEL-beanng proteins to ER fl Recycling of" Q +9” final: ma K. L ,9 KDEL a i ERGIC (VTC) onNard l.|. EFI-to Golgi Ira nap-on VESICIB KDE L receplur Hoth ER The KDEL-Receptor (KDELR), a 7TM molecule / Cg) —b To medial Golgi Secreted protein without KDEL sequence KDEL KDEL {Cis Golgi Netw. KDELR ,. e 09} 3 In the Golgi or VTC the KDELR picks up ER- resiclent molecules and interacts with COPI. The KDELR is an ER transmembrane protein with the C-terminal KKXX motif. Relocation to the ER involves phosphorylation. In the ER this receptor uses another motif FFXXBB(X)rl to bind COPII molecules. | . KKXX motif. enables COPI coating FFXXBBIXIII motif. enables COPII coating KDEL-binding Elle Lumen Cytopl. pH environments in the ER and Golgi apparatus pH . neutral gradient vesicular KDEL ER resident protein cis Golgi stack trans Forward pathway = secretory pathway = anterograde pathway = bulk pathway MANNOSE TAGGING FOR TRANSPORT BY LYSOSOMES Return pathway = recycling pathway = retrieval pathway = retrograde pathway /Jvlan Protein M5“ moiety / \Man ——--- Man lcNac ------ GlcNac ----- Man \\ Man ----- Man ----- Man 2 UDP-GICNAC N-Acetylg'ucosamine Phosphotransferase 2 5-P-Gl N Man/l - C so )llan< IcNac ------ GlcNac ----- Man\ Man Man Man .. Man ____ Man E—P—Glcmac 2 GICNAC N-Acetylglucosammy' 1'Phosphodlester o.- N-Acetylglucosaminidase / \Man ----- Man GlcNac ------ GlcNac ----- Man Manuman Replaces 10.29 M GRADUAL CHANGES INTRODUCED T0 GLYCOPROTEINS AS THEY TRAVERSE THE GOLGI APPARATUS O “.4. . mm 0“ A ocxl-(i 1 dilatiode d —i.| uer Hyuid 0 _ SORTING let's Golgi _ . phosphorylation of lnetwork a H "m" oligosaccharides on ” am” i | sosomalproteins - Endoglycoswase H 0H4 «or Jupu d we lgggtema SEnSiTiVe e-xl-‘l . removal of Man J Dfipmannou V o removal of Man :nf'etd'al - Madial . addition of GlcNAc CIS erna 9‘36“ . addition of Gal ’1 trans - addition of NANA ‘cistema Gal 2| stack . sulfation of tyrosines and carbohydrates trans Golg' TGN SOHTING lnetwlo’k Endoglycosidase H , resistant plasma secretory membrane vesicle lysosome Endo-H [endoglycosidase H] cleaves off high mannose and some hybrid oligosaccharides of N-linked glycoproteins. The secretory pathway and the regulated secretory pathway Constitutive Induced ER Baseline protein synthesis. _ - Increased protein synthesis. Maintenance molecules are produced. 60'9" - New molecules are produced. - Vesicles containing \ / molecules with signal patches. Lysosomes Plasma membrane A Signal patch is a Secretion (extracellular matrix) molecular conformation determining a specific modification that targets a molecule for a certain destination. VESICLE COATING and TRANSPORT Clathrin-coated vesicles: external uptake via endocytosis. Also. transport from the trans Golgi network in lysosomes. COPI-coated vesicles (coat protein complex type 1): recovery by the ER (retrograde transport) of lumenal molecules with KDEL or transmembrane molecules with KKXX C-terminal sequences previously exported to the VTC or Golgi. COPll-coated vesicles (coetprotet’n complex type II): from the ER. along the secretory pathway, to the Golgi. E ) COPII COPI CLATHRIN CLATHRIN lysosomes plasma membrane VESICULAR TUBULAR CLUSTERS As soon as the vesicle tubular clusters form, they begin budding off vesicles of their own. Unlike the COPll-coated vesicles, which bud from the ER, VTC- derived vesicles are coated with COPI. Vesicles from the VTC or cis-Golgi carry ER resident proteins that have “escaped” back to the ER (retrograde pathway). They also carry back proteins that participated in the ER budding reaction so they can function again (recycling). ' 9 MOLECULAR TRAFFIC BETWEEN THE ER, GOLGI, How are cargo and coat molecules recruited. ENDOCYTIC PATHWAY, AND PLASMA MEMBRANE gems...“ m. ENDOPLASMIC RETICULUM LYSOSOME adaptors 5e024 Sec23 g _ proximal coat proteins cargo proteins E.R. lumen 1. Recruitment of a small G protein (Sar1, a GTPase) to the membrane. This process is mediated by its specific GEF. 2. Small G protein molecules recruit coatamers (COPII on the ER). 3. Coatamers selectively bind cargo receptors. Coat assembly leads to soluble cargo recruitment and membrane curvature (vesicle formation). EARLY ENDOSOME Recycling Plasma membrane CELL EXTERIOR Based on: C'elf116: 153. 2004. Cell 122: 606, 3005, andfiiofec. C‘eflm’arBz'ol. 26: 8011. 2006 GENERAL MECHANISMS OF VESICLE TARGETING AND DOCKING IN THE ER AND GOLGI APPARATUS - The assembly of coat proteins (COPI, COP", ciathrin) around vesicles is driven by molecules such as ARF or Sar-1 in a GTP-dependent manner. 0 Dissociation of the coat occurs when the vesicle is pinched off and protein- GTP is hydrolyzed to protein-GDP by a membrane-associated GAP. - Coat assembly resumes when GTP replaces GDP with the assistance of a GEF. ‘ Vesicle fusion with its target is regulated by a N-ethylmaleimide-sensitive protein factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). - v-SNAREs on vesicles engage t-SNARES on the corresponding target membrane. SNAP binds v-SNARE-tSNARE complex NSF binds SNARE-SNAP complexes SNAP recruits NSF to SNARE complexes, to initiate SNARE detachment via ATP hydrolysis. VESICLE FUSION 1) The assembly of coatomers around vesicles is driven by molecules such as the ARF or Sar-1 in a GTP-dependent manner. 2, 3) Vesicle buds off. GTP is hydrolyzed by a membrane- associated GAP (AR FGAP, Sam-GAP). Dissociation of coatomers follows. A new coat could be assembled after a GEF enables replacement of GDP with GTP (ARFGTP and Sar1GTP regenerate). 4. 5) V— and t-SNARES engage each other. Fusion starts. 6) N-ethyI-maleimide—sensitiye protein factor (NSF) and soluble NSF attachment proteins (SNAPs) cause the SNAREs to disengage. completing fusion. 7) NSF (an ATPase) and SNAP are # released. y—SNARE and t—SNARE can be re—used in new fusions. Donor membrane - ARF or Sar1 . |: v-SNARE I t-SNARE ARFGAP or Sar1 GAP ARF-GDP or ‘- Sarl-GDP \ I. 3 . n‘ container “recychng NSF 6 Stages ofvesicle fusion: budding, coating, scission, uncoating, tethering, docking, and fusion. m NSF — y-SNARI: 2 GTPDD szARE 4 l t-SNARE 3. 4 CD vSNARE I % FUSION ‘- BEGINS 'Q E E 5 v—SNARE_ 1-. CD CD h m ('5 ecruitment '— t-SNARE at: NSF aw“ afar, m] p3? we” t-SNARE D°\Q\e 7 Cr CELLULAR VESICULAR TRANSPORT Extrusion Recycling Pinocytosis. RME or Phagocytosis a; gag!» O Q “ , + . at g g Fusron to cell membrane USIOn to cell membrane a; O 0 Endosome formation V..__Rab11, Rab1? §Rab5A, B, c o '- LE * o Transport vesicle 3 "‘4 iv |IIIIIIIIIIIIIIIIIIBNIIIIIIIIIIIIIIII|| ExocytOSiS TGN: Rabfi, RabB '- Rab13 [secretion] Medial stacks: Robs, Rab12 t secre or}.r CGN: Rab24 apparatus 22:: vesicles It 3‘ -- ' Rab3A Rab1A, B, Rab2 Transport vesicle ER RME: receptor-mediated endocytosis. CURL: compartment of uncoupling of receptor and ligand. for RME. EE: early endosome. LE: late endosome. CGN: cis Golgi network. TGN: trans Golgi network. Rabi: members ofthe Rab family of small G proteins. ...
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This note was uploaded on 06/02/2008 for the course MMG 409 taught by Professor Arvidson during the Summer '08 term at Michigan State University.

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CH10_HO - ENDOPLASMIC RETICULUM An organelle adjacent to...

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