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Beyond the central dogma Central dogma culminates with synthesis of protein in cytoplasm But can’t mix proteins, polysaccharides, lipids and nucleotides together and get a living cell Formation of a cell requires the context of a pre-existing cell Cell structures (organelles; mitochondria, chloroplasts, Golgi, ER) and organization must be inherited, just like DNA Epigenetics Lecture 14 cont’d Intro to protein import into organelles Signal sequences Import into the nucleus Import into mitochondria and chloroplasts Import into ER, vesicle trafficking Note - in the next few lectures I will show many figures from Molecular Biology of the Cell 4th ed. (Alberts et al.) On reserve at Marriott Nuclear import occurs through pores in the double membrane “Nuclear envelope”= Outer nuclear membrane ECB 15-7 Inner nuclear membrane Perinuclear space ER Nuclear pores All nuclear transport occurs through nuclear pores What molecules must be imported into nucleus? Exported? Nuclear lamina Nuclear pores are large protein complexes Cytplasmic fibrils Cytosol Cytoplasmic face Nuclear envelope Nucleus Nuclear lamina Annular subunit of central channel or transporter Nuclear basket or cage Nuclear face ECB 15-8 Multiple copies of ~100 different proteins (nuclear pore proteins = NPPs) totaling >125 million daltons! Transport of large molecules is active requires GTP Small molecules (< 60 kDa), or about 9 nm diameter) enter or exit nucleus by passive diffusion Nuclear pores also required for active export of RNPs (including ribosome subunits, mRNA, tRNA etc.) Larger molecules must be actively tranported: (1) binding to transporter; and (2) transport thru nuclear pore using GTP Import and export occur through same pores A nuclear localization signal (NLS) is necessary and sufficient for nuclear import of proteins The “classical” signal for nuclear import includes multiple basic amino acids (K = lysine and R = arginine)…example P-P-K-K-K-R-K-V NLS can be anywhere in protein sequence Simplified view of nuclear transport NLS (cargo) (importin) Pore opens ECB 15-9 Energy for transport provided by G proteins (GTP binding proteins; large family) Molecular “switches” Pi GAP GDP GTP GTPase “on” GTP GEF GTPase “off” GDP GAP = GTPase Activating Protein GEF = Guanine Nucleotide Exchange Factor Pi RAN GTPase used in nuclear transport GAP GDP GTP GTPase “on” GTP GEF GTPase “off” GDP Nuclear import/export cycle is driven by GTP hydrolysis Q: relation to protein transport?? Directional protein import is driven by GTP hydrolysis NLS Importin Importin Pi + NLS Ran GAP Cytoplasm Ran-GTP Importin Ran-GDP Importin (NLS receptor) binds cargo (with NLS) in cytoplasm Importin-cargo transported into nucleus thru nuclear pore Ran-GTP in nucleus binds importin, importin releases NLS (cargo) Ran-GTP-importin exported from nucleus thru pore Ran-GAP stimulates GTP hydrolysis in cytoplasm by Ran Ran-GDP releases importin in cytoplasm Nucleus NLS Importin Importin Ran-GTP GTP GDP Ran-GTP Ran-GDP RanGEF NLS Ran-GDP transported into nucleus (not shown) Ran GEF stimulates nucleotide exchange restoring Ran-GTP. Specific signals direct export from the nucleus: lessons from HIV Human immunodeficiency virus (HIV) is a “retrovirus:” Human T lymphocyte Transport DS vDNA RNA genome with DS DNA intermediate Reverse transcription vRNA Integration Uncoating RNA is “reverse transcribed” to make DS DNA Unspliced vRNA is trapped in nucleus (contains introns-no export) Transcription Unspliced vRNA (9 kB) HIV Processing GpppC mRNA (2 kB) Progeny virus exits host cell by budding Alternative splicing produces over 30 mature mRNA that are exported and translated AAA Nucleus Rev Transport Translation Cytoplasm Nuclear Export Signal Rev is req’d for export of RevvRNA from nucleus One protein, Rev contains a NLS and is tranported into the nucleus Rev binds “Rev-response element” on vRNA Rev-RNA complex exported, RNA packaged and virus leaves cell Lecture 14 Intro to protein import into organelles Import into the nucleus Import into mitochondria and chloroplasts Recall mitochondrial and chloroplast structure Organelle DNA - encodes small % proteins, human mitochondria encode only 13 proteins Rest (thousands) encoded in nucleus, transcribed, exported to cytoplasm, translated and imported into correct organelle And correct compartment in that organelle Import into mitochondria is post-translational Translate mRNA for mitochondrial matrix protein in vitro Proteins contain Nterminal “signal sequence” Trypsin Add “energized” mitochondria Protein imported into mitochondria Trypsin Imported matrix protein is protected from added protease Digest with protease Protein degraded Import into mitochondria and chloroplasts is post-translational Import is directed by a signal sequence at the Nterminus of mitochondrial proteins Non-polar aa (green) on the other… Positive charge (red) clustered on one face of helix… No conserved sequence Predicted to form “amphipathic” a-helix Cleaved after protein is imported MBoC (4) figure 12-23 © Garland Publishing TOMs and TIMs: Import into the mitochondria matrix requires two membrane transporters… Transport thru aqueous channels: “TOM” and “TIMs” (Translocaters in Outer/Inner Membrane) Mitochondrial import signal binds receptor in outer membrane (assoc w “TOM”) “Contact site” (close apposition of OM & IM) Cytoplasm Outer membrane Intermembrane space Inner membrane “TIM23” Matrix protein w N-terminal signal sequence Import receptor “TOM” Removal of signal sequence Matrix Mature matrix protein See ECB figure 15-10 Protein import into mitochondria requires energy… Cytosolic HSP70 ADP + Pi ATP Cytoplasm Outer membrane TOM IMS ++++++ Inner membrane Matrix ---- +++++++++ TIM23 ---------- Adapted from MBoC (4) figure 12-27 © Garland Publishing ATP Mitochondrial HSP70 ADP + Pi (1) Electrical potential (DY) across inner membrane req’d to initiate transport (2) Cytosolic HSP70 unfolds protein for import (ATP used) (3) Mitochondrial HSP70 refolds protein after import (ATP used) How are proteins targeted to other mito membranes/compartments? ../L14OrganelleImport/15.5-mito_import.mov How are proteins targeted to mitochondrial membranes and compartments? …the direct route Cytoplasm TOM Protein in IM Protein in IMS Outer membrane IMS Inner membrane TIM Matrix “Stop transfer” signal Cleaved stop transfer (degraded) Matrix signal (cleaved and degraded) Adapted from MBoC (4) figure 12-29 As before, signal sequence directs import through TOM/TIM23… “Stop transfer” signal interrupts translocation through TIM23, releasing protein to inner membrane… Cleavage of stop transfer signal releases protein to intermembrane space… Import into the thylakoid requires multiple signals Transporters Cytosol Outer membrane Transit peptide IMS Inner membrane Receptor Transit peptide cleaved Stroma Thylakoid signal Thylakoid A “transit peptide” (an amphipathic helix) targets to chloroplast stroma (similar to mitochondrial signal peptide, but NOT interchangeable!) Evidence for four paths to thylakoid Adapted from MBoC (4) figure 12-30 © Garland Publishing Protein targeting NLS: (basic) Protein targeting Nucleus NES: (L-rich) Mitochondria Signal peptide Cytoplasm Additional signals for subcompartments Chloroplasts Vesicle targeting RER See ECB figure 15-5 Golgi Endosomes Secretory vesicles Plasma membrane Transport Retrieval Next two lectures Lysosomes L15: Protein and vesicle targeting Today - import into ER, begin vesicle targeting ECB 15-5 ER network is extensive GFP-protein in plant cell ER TEM of RER in dog pancreas Note ribosomes on membrane Vesicles derived from ER by biochemical prep are termed microsomes Some ribosomes bind to ER ECB 15-12 What is the evidence for cotranslational transport? Transport of protein into ER is cotranslational Translate mRNA in vitro… In vitro product ~2 kDa larger than in vivo product ~15-25 addtnl aa at N-terminus Add protease - product degraded Add RER microsomes AFTER translation… Product still ~2kDa larger than in vivo product… Add protease… Product degraded… Add RER microsomes DURING translation… Product processed to mature form… Add protease… Product protected… INSIDE microsomes! The “Signal Hypothesis” From results of experiments such as these, Dobberstein and Blobel proposed a hypothesis 1. The signal for translocation of a secretory protein into the ER resides in the nascent polypeptide, in the form of a leader “pre-” sequence or “signal peptide;” 2. Translocation of the polypeptide across the ER membrane is co-translational (unlike import into nucleus, mito, and chl); and 3. the signal peptide is cleaved post-translationally in the ER lumen by a “signal peptidase.” Blobel - Nobel prize 1999 ER Signal Sequence No conserved sequence Signal sequence is 12-25 amino acids Predicted to form a-helix with hydrophobic core (yellow aa above) Signal sequence is both necessary and sufficient for import into ER Necessary Sufficient Requirements for targeting and translocation into the ER 1. “Signal sequence” : hydrophobic a-helix in nascent protein 2. “Signal recognition particle (SRP):” cytoplasmic complex of protein and RNA binds signal sequence 3. “SRP-receptor:” integral ER membrane protein 4. “Translocon:” an aqueous channel through ER membrane (sec61 complex) Targeting to RER ECB 15-13 1. Translation exposes signal sequence outside ribosome 2. SRP -a complex of 300bp RNA and 6 proteins- binds the signal sequence in nascent protein, transiently arrests translation 3. SRP-arrested ribosome binds SRP receptor in ER membrane (targeting) 4. Ribosome and polypeptide handed to a translocation channel (“translocon”). SRP and SRP-R are recycled (requires GTP hydrolysis). Translation resumes and translocation begins Proteins destined for secretion enter ER lumen ECB 15-14 Signal peptide targets nascent protein to RER as before Signal peptide is cleaved by signal peptidase associated with translocation channel Translation and translocation are completed, releasing completed polypeptide into lumen of RER Signal peptide is degraded What about membrane proteins?? Membrane proteins contain stop transfer sequence ECB 15-15 As before, signal peptide targets nascent protein to RER However, “Stop transfer” sequence halts translocation Protein is released from translocon Stop transfer sequence acts as transmembrane domain Double- and multipass membrane proteins ECB 15-16 Internal signal sequence targets nascent protein to RER… “Stop transfer” sequence halts translocation and releases protein from translocon… Signal sequence and stop transfer sequence act as transmembrane domains Protein folding in the ER is assisted by “BiP”… “Binding protein” (HSP70 family of ATPases) in ER lumen binds nascent polypeptide as it is being translocated, and assists folding (and translocation?)… C N Signal peptide Signal peptide N RER membrane ER Lumen Translocon (sec 61 complex) Adapted from MBoC (4) figure 12-46. See ECB figure 15-14 BiP ADP+Pi Signal peptidase ATP BiP BiP binds nascent protein during translation/translocation… C N “Secreted protein” in lumen of RER Release of BiP from folded polypeptide requires energy (ATP)… Incorrectly folded proteins are held in ER until folded properly, or are targeted for degradation… The topology of a membrane protein can be predicted… Hydropathy plot for Rhodopsin 1 2 3 4 5 6 Topology of Rhodopsin 7 COOH Hydrophobic A B C D Cytoplasm 1 2 3 4 5 6 7 Hydrophilic ER Lumen NH2 Adapted from MBoC (4) Figure 12-50 © Garland Publishing Hydrophobic a-helices of 15-25 aa are predicted to be membrane spanning domains…and also function as “topogenic sequences.” Seven domains in rhodopsin 100 200 “Start transfer” initiate protein translocation, “Stop transfer” sequences halt translocation… Note start sequences can be in either orientation H2N- 1 Start A 2 3 Start Stop B 4 5 Start Stop C 6 7 Start Stop D -COOH Review of the “Signal Hypothesis” 1. The signal for translocation/insertion of a protein into the ER membrane resides in the nascent polypeptide, in the form of a “signal sequence.” 2. Translocation of the polypeptide across the ER membrane is co-translational 3. The signal peptide (of secreted proteins) is cleaved post-translationally in the ER lumen by a “signal peptidase.” 4. Four components: (1) signal sequence, (2) SRP, (3) SRP-R, and (4) translocon 5. Uncleaved signal sequences (and “stop transfer” sequences) function as transmembrane domains in integral membrane proteins… 6. The topology of a protein can be predicted from the “hydropathy” plot of its amino acid sequence… 15.7-ERprotein_trans.mov Vesicle targeting Protein and vesicle targeting NLS: (basic) Protein targeting Nucleus NES: (L-rich) Mitochondria Signal peptide Cytoplasm Additional signals for subcompartments… Chloroplasts Vesicle targeting RER See ECB figure 15-5 Golgi Endosomes Secretory vesicles Lysosomes Plasma membrane Transport Retrieval 15.1-cell_compartments.mov Membrane cycling endocytosis ECB15-17 Exocytosis (secretion) Secreted proteins Plasma membrane proteins Transport is highly regulated so vesicles carry appropriate cargo for their specific destination Lumen of organelle is equivalent to outside of cell x x x Begin with ER to Golgi x x x What about membrane protein in ER? Modification of proteins begins in ER Disulfide bridges Glycosylation - ECB 15-22 common in plasma membrane and secreted proteins Asn-X-Ser Most common glycosylation is addition of a specific oligosaccharide (14mer) to asparagine during translation. Addition is to the NH2 group; N-linked glycoproteins Addition is done in a single step by transfer from specialized dolichol lipid This oligo is then extensively modified in diverse ways Modification begins in ER: Transported to Golgi for more processing From the ER, proteins are transported to the Golgi Nuclear envelope RER Vesicular-tubular clusters to CGN Golgi MBoC (4) figure 13-22 © Garland Publishing Proteins leave the ER in transport vesicles budding from exit sites… Transport vesicles from ER fuse to form vesicular-tubular clusters… Vesicular-tubular clusters enter the Golgi by fusing with the cis-Golgi network (CGN) Glycoproteins are “processed” as they pass thru the Golgi… From the ER, proteins are transported to the Golgi cis-Golgi network (CGN) Vesicular-tubular clusters in from RER… cis medial trans Trans Golgi network (TGN) ECB 15-24 Proteins leave the ER in transport vesicles budding from exit sites… Transport vesicles from ER fuse to form vesicular-tubular clusters… Vesicular-tubular clusters enter the Golgi by fusing with the cis-Golgi network (CGN)… Glycoproteins are “processed” as they pass thru the Golgi… The Golgi is biochemically compartmentalized… Nucleotide diphosphatase (trans) Osmium (cis) MBoC (4) figure 13-28 © Garland Publishing Acid phosphatase (TGN) Glycoproteins are further processed in the Golgi Protein synthesis ER Golgi apparatus CGN cis Removal of mannose Addition of GlcNAc Glycosylation at H3N+…XXNXSXX…COOAs protein moves through Golgi, monosaccharides are added or removed in specific Golgi compartments medial GlcNAc = N-acetylglucosamine Mannose Glucose Addition of galactose trans TGN Lysosome Plasma membrane Constituitive secretion (Default?) Regulated secretion Secretory vesicles Fucose Galactose, etc. Proteins are sorted in the TGN… Constitutive secretion… Regulated secretion… Lysosome… Why are membrane/secreted proteins glycosylated? Structure and folding? Protection of cell (protein) from external proteases? Function? adhesion… signaling… The plasma membrane of many (most?) cells is coated with glycoproteins Transport through Golgi cis-Golgi network (CGN) Vesicular-tubular clusters in from RER… 1. Vesicle transport 2. “Cisternal maturation” “Budding” cis Transport vesicles “Fusion” medial trans trans-Golgi network (TGN) ECB 15-24 Transport vesicles out Cisternal maturation and vesicle transport probably both contribute to membrane flow through Golgi Next time Vesicle transport from ER to Golgi Transport from Golgi Constitutive secretion Regulated secretion To lysosome