Moving Proteins into Membranes & Organelles PDF

Moving Proteins into Membranes & Organelles  Protein Sorting how proteins are targeted to & translocated across the membranes of different cellular organelles a typical mammalian cell contains thousands of proteins – may be localized to: the cytosol wit...

Moving Proteins into Membranes & Organelles  Protein Sorting how proteins are targeted to & translocated across the membranes of different cellular organelles a typical mammalian cell contains thousands of proteins – may be localized to: the cytosol within organelles embedded in organellar membranes – may also be meant for export from the cell or for positioning in the plasma membrane  Protein targeting or sorting Two processes are involved 1. signal-based targeting 2. vesicle- based trafficking - For membrane proteins, targeting leads to insertion of the protein in the lipid bilayer -For water soluble protein, targeting leads to translocation of the entire protein across the membrane into the aqueous interior of the organelle  How are Proteins Sorted? Two general, but different, processes are involved: 1. Nonsecretory Pathway: Targeting to organellar membranes & transport across organellar membranes – refers to the sorting of proteins synthesized on free ribosomes – localized to the cytosol, peroxisomes, mitochondria, chloroplasts & nucleus 2. Secretory Pathway: Targeting to the plasma membrane & export from the cell – refers to the sorting of proteins synthesized on bound ribosomes – these proteins may be excreted, localized to the ER, plasma membrane, Golgi apparatus or to lysosomes Eukaryotic Protein-sorting Pathways Non secretory pathway  How are Proteins Targeted? information needed to target proteins to particular organelles is encoded within the protein in a sequence of about 20-50 amino acids – called targeting sequences; these are also called signal sequences or signal peptides each organelle carries receptors that bind to only one type of signal sequence bound receptor then transfers the polypeptide to a translocation channel protein is then passed through the lipid bilayer, by coupling the translocation to an energetically favorable process example: ATP hydrolysis Four Considerations in Protein-Targeting Mechanisms 1. What is the nature of the signal sequence and what makes it different from other signal sequences? 2. What is the receptor for the signal sequence? 3. What is the structure of the translocation channel, and do folded or unfolded proteins pass? 4. What is the source of energy that drives unidirectional transfer across the membrane?  Secretory Pathway Soluble, secreted proteins whose final destination is localization to the plasma membrane, lysosomes, or secretion from the cell use the secretory pathway  Signal Sequence Targets Nascent Secretory Proteins to the ER After synthesis begins on free ribosomes in cytosol, 16-30 aa ER signal sequence directs ribosome to ER membrane (making it RER) ER Signal sequence: – found at N-terminus – typically cleaved before mature protein formed, often prior to separation of polypeptide from ribosome – contain >1 + charged aa next to a core of 6 -12 hydrophobic aa – hydrophobic core critical for binding to receptor and translocation  Co-translational Translocation translocation of the nascent polypeptide occurs before the protein is fully synthesized fully formed protein cannot enter the ER typically a ~70 aa long sequence (including signal sequence) is formed before translocation begins mechanism of cotranslational translocation requires 2 components: Signal-Recognition Particle (SRP) SRP Receptor on the ER membrane  Signal-Recognition Particle (SRP) SRP consists of: 300 nt RNA molecule bound to 6 different polypeptides (P54, P19, P68, P72,P9 & P14) role of RNA is not fully understood – may serve as scaffold to support the proteins – May also have an active role in the process P54 recognizes and binds to the nascent protein via the signal sequence P9 & P14 bind to the ribosome P68 and P72 are required for translocation P19 is required for attachment of P54 to the rest of the particle  SRP Receptor Integral membrane protein Consists of 2 subunits: large α subunit & smaller β subunit – α subunit interacts w/ the SRP & binds GTP – β subunit is embedded in the membrane  Translocon: translocation channel analogous to chemical-gated channels (transmembrane protein) consists of the Sec61 complex of proteins closed when it is not bound to SRP/SRP receptor/ribosome complex open once it binds to the complex permits signal sequence (& polypeptide) to enter signal sequence comes in contact w/ aa of the Sec61 complex Co-translational Translocation Across the ER membrane  Post-translational Translocation Most eukaryotes: co-translational translocation used for secretory proteins to enter ER In yeast (and other eukaryotes) some proteins enter ER lumen after translation has completed Unidirectional translocation involves: – Sec61 translocon – Sec63 complex – BiP (chaperone) – do not use the SRP or SRP receptor  Export of Bacterial Proteins Yersinia pestis is a Gram negative bacteria that causes bubonic plague injects disabling proteins into macrophages by type III secretion apparatus (T3SS) incapacitated macrophages cannot destroy the bacteria, thus permitting them to increase in numbers  Membrane Proteins: Insertion of Proteins into the ER Membrane Each membrane protein has a unique orientation with respect to the phospholipid bilayer – ER, golgi, lysosomes, plasma membrane Integral proteins that are synthesized in RER remain embedded in membrane as they move to final destination – Orientation along the way is preserved – ie: some segments face cytosol, other face opposite direction all the way through Therefore, orientation of membrane proteins are determined in the ER membrane Topology of an integral membrane protein refers to: – the orientation of its membrane-spanning segments – the number of such segments  ER Membrane Proteins Integral proteins are categorized in topological classes – Types I, II and III are single-pass proteins – Type IV are multi-pass proteins – GPI linked proteins are tethered to membrane w/ phospholipid  Type I Proteins N-terminal signal sequence, that is cleaved N terminus in exoplasmic face & C terminus in cytosol stop-transfer anchor sequence: – 22aa hydrophobic sequence that stops translocation – lateral movement across translocon wall inserts protein in membrane  Type II Proteins lack a cleavable N-terminus contain a signal-anchor sequence – acts as both ER signal sequence & membrane- anchor sequence N terminus faces the cytosol  Type III Proteins lack a cleavable N-terminus contain a signal-anchor sequence that acts as both an ER signal sequence and a membrane-anchor sequence N terminus faces the exoplasmic face  Type IV Proteins Type IV A Proteins N-terminus in the cytosol Examples: – the GLUT transporters – most ion channels, including ABC superfamily transporters Type IV B Proteins N-terminus extends into the exoplasmic face Examples: – the G-protein coupled receptors Arrangement of Topogenic Sequences in ER Membrane  Lipid-anchored Proteins final class of membrane protein do not have hydrophobic membrane-spanning domains instead are anchored by amphipathic phospholipids anchoring molecule is GPI glycosylphosphatidylinositol – called GPI linked proteins  Protein Modifications Proteins may be modified in the ER, Golgi & secretory vesicles Four principal modifications before they reach their final destination: Glycosylation in the ER & Golgi Formation of disulfide bonds in the ER Proper polypeptide folding & assembly of multi subunit proteins in the ER Proteolytic cleavages in the ER, Golgi & secretory vesicles  Glycosylation Principal chemical modification to most proteins Carbohydrates may be added to: – the OH group of serine and threonine named O-linked oligosaccharides – the NH2 group of asparagine most common Named N-linked oligosaccharides Why glycosylation? – Promotes proper folding – Promotes stability 13-16: N-linked oligosaccharides Addition & Initial Processing of N-linked Glycosylation in ER  Disulfide Bonds these covalent bonds help stabilize tertiary & quaternary structures Protein Disulfide Isomerase (PDI) – Enzyme that catalyzes addition of disulfide bonds – found in the lumen of the ER of all eukaryotic cells Only proteins that enter the lumen of the ER contain disulfide bridges: – disulfide bonds only found in secretory proteins & exoplasmic domains of membrane proteins – proteins synthesized on free ribosomes are stabilized by other means Formation & Rearrangement of Disulfide Bonds by PDI  Folding Folding is facilitated by chaperones and other ER proteins. – Lectins are carbohydrate-binding proteins Bind selectively to N-linked glycosylated proteins eg) calnexin, calreticulin – Peptidyl-propyl isomerases Enzymes that accelerate rotation about peptidyl- prolyl bonds in unfolded segments What about Improperly Folded Proteins? Misfolded proteins cannot exit the lumen of the RER Activation of the unfolded protein response: – Accumulation of unfolded proteins within the lumen of the RER will cause the synthesis of proteins that assists folding – Ire1: ER membrane protein that exists that is a key participant in the unfolded protein response – Hac1: TS factor that turns on TS of genes encoding several protein-folding catalysts  Hereditary (Familial) Emphysema Illustrates detrimental effects that can result from misfolding of proteins in the ER Hereditary point mutation of a blood component called alpha-1-antitrypsin (AAT) – normally secreted by hepatocytes & macrophages – absence results in the loss of a lung structural protein, elastin. – The mutant AAT does not fold properly and is not exported from the cell – Mutant AAT can not inhibit elastase, resulting in destruction of elastin Wild type AAT binds to and inhibits both trypsin & elastase – Elastase breaks down elastin  Degradation of Misfolded Proteins misfolded proteins may also be degraded they are exported through the translocon to the cytosol typically broken down by ubiquitin/ proteasome associated pathways Figure 3-29: Ubiquitin & proteasome mediated proteolysis  Sorting of Proteins to Mitochondria Mitochondrial proteins may be synthesized on ribosomes found in the mitochondrial matrix, but the majority of the proteins used in mitochondria are synthesized on free ribosomes in the cytosol and imported Mitochondrial destined proteins from cytosol have targeting sequence named MTS: – Found at N-terminus – 20-50 aa long – Amphipathic: R & K aa on one end; hydrophobic aa on the other – MTS is cleaved in the matrix Import into the Mitochondrion: – only an actively respiring mitochondrion can import proteins proton-motive force is required for translocation – only unfolded proteins can cross mitochondrial membranes  Mitochondrial Import protein maintained in unfolded state by bound Hsc70 chaperone targeting sequence binds to Tom20/22 – OMM import receptor protein transferred to Tom40 – OMM general import pore protein binds to Tim23/17 – IMM pore translocated to matrix at contact sites b/w IMM & OMM protein w/ matrix chaperone Hsc70 localized to pore w/ Tim44 once protein is in matrix, signal sequence cleaved by MPP final protein folding requires help of matrix chaperonins Targeting to Sub-Mitochondrial Compartments Proteins have different signal sequences & pathways that target them to different mitochondrial areas  Peroxisomal Proteins Peroxisomes: single-membrane bound organelles that contain oxidases and catalase all peroxisomal proteins are synthesized on cytosolic free ribosomes Peroxisomal proteins are imported in a folded state Most require a C-terminal target sequence called PTS1 (peroxisomal targeting sequence) – PTS1 contains a SKL (serine-lysine-leucine) motif A few require N-terminal PTS2 requires ATP Mechanism of Translocation into Peroxisome Protein with PTS1 sequence binds to a free cytosolic receptor, Pex5. Bound Pex5 interacts with the membrane-bound receptor Pex14. Bound Pex5 is then transferred to a complex of transmembrane proteins that induce translocation. Unbound Pex5 is returned to the cytosol. PTS2 uses Pex7 instead  Zellweger Syndrome rare, congenital disorder Autosomal recessive mutation involving defective peroxisomes Transport of peroxisomal proteins is impaired, peroxisomes are fewer in number and do not work Lead to severe impairment of many organs – liver, kidneys & brain Common features include: – enlarged liver, prenatal growth failure – high blood levels of iron & copper – vision disturbances – at birth: lack of muscle tone, an inability to move (suck, swallow), unusual facial characteristics, mental retardation, seizures, jaundice & GI bleeding No cure or treatment for Zellweger syndrome – Most infants do not survive past first 6 months – usually succumb to respiratory distress, GI bleeding or liver failure  Transport Across the Nuclear Membrane Nucleus surrounded by two membranes: each membrane consists of phospholipid bilayer and proteins. Molecules of all sizes enter and leave the nucleus via Nuclear Pore Complexes (NPC) NPCs are large allowing for bidirectional transport: – passive diffusion of small molecules, ions – selective energy- dependent transport of nuclear proteins, RNAs, and RNPs (>9nm)  NPC Structure NPC composed of different nucleoporin proteins attached to nuclear basket on nuclear side, cytoplasmic filaments on cytoplasmic side, and central transporter through the middle  FG Nucleoporins and Transporters FG nucleoporins contain many Nuclear transporters short hydrophobic FG repeats and have hydrophobic long hydrophilic regions regions on their surface (dark blue dots) that bind reversibly to FG-domains in FG-nucleoporin. Form molecular sieve that allows H2O-soluble molecules through, but not macromolecules FG = phenylalanine and Glycine  Import into the Nucleus Proteins synthesized in the cytosol and meant for use in the nucleus enter via the NPC Such proteins contain a nuclear-localization signal (NLS) that directs their translocation into nucleus – NLS is a short amino acid sequence often near the protein’s C-terminus Importins are the transport proteins that bind the NLS and transport the NLS containing cargo into the Immunofluorescence shows fusing a nucleus NLS to a cytoplasmic protein causes the protein to enter the nucleus. Mechanism for Nuclear Import Free cytosolic importin binds NLS of cargo protein Complex moves through NPC, interacting w/ nucleoporins Once in nucleoplasm, conformational change of importin (via interaction w/ Ran-GTP) results in lower affinity for NLS and release of cargo Transporters are recycled  Export out of the Nucleus Similar mechanism used to export proteins, tRNAs and ribosomal subunits from the nucleus to the cytoplasm. Exportin: transport cargo proteins containing nuclear-export signals (NES) out of the nucleus Export mechanism: In nucleoplasm, exportin binds to NES of cargo protein and Ran-GTP Complex diffuses through NPC via interactions w/ nucleoporins Dissociation from complex after Ran-GTP hydrolysis Transporters recycled  Summary: Import & Export of Proteins through NPC Cargo proteins bear an NES or NLS, translocate thru NPC – Some proteins shuttle b/w nucleus & cytoplasm, and must have both NLS & NES Both processes require Ran, a G protein that exists in different conformations when bound to GTP or GDP  GTP Switch Proteins Guanine nucleotide-binding proteins: – switch “on” when GTP bound – switch “off” when GDP bound Signal induced conversion of inactive to active state is mediated by GEF, releasing GDP, allowing GTP to bind – GEF: guanine nt exchange factors Conversion from active to inactive form by hydrolysis of GTP, accelerated by GAP – GAP: GTPase-activating proteins Fig 3-32