Unit 5 Flashcards
Relative volumes occupied by major membrane-enclosed organelles in liver cell
- Mitochondria: 1700/cell
- ER: 12%
- Golgi: 3%
- Vesicles: 1% and 200-400 per type
Origin of mitochondria
Anaerobic euk cell w/ membrane bound nucleus + ER —> engulfed aerobic bacterium
Evolution of nuclear + ER membranes
- Precursors of euks believed to be organism (like bacteria) with no internal membranes
- Plasma membrane carried out all membrane-related functions
- Endomembrane system evolved as invagination of plasma membrane
- Mitochondria + chloroplasts evolved as endosymbionts
Three ways to import proteins into organelles
- Through nuclear pores
- Across membranes
- By vesicles
- Protein sorting = transfer or proteins into compartments where they are needed
- Synthesis of virtually all proteins starts in cytosol on free ribosomes
- All protein transport requires energy
Signal sequence
- Stretch of AAs (15-60 AAs long) –> directs proteins to particular organelles (for nucleus, mito/chloro, peroxi, ER)
- Usually removed after sorting
- Delete or transfer sequence to another protein –> protein goes to wrong “address”
Nuclear envelope
- Double membrane of nucleus
- Close association w/ ER
- Nuclear lamina: strand material inside (where genetic material is)
- Nuclear pores: allow proteins into nucleus
Nuclear pore complex
- Nuclear basket
- Gateway proteins block passage
- Very high traffic but highly selective (500 molecules through each of 3000-4000 pores/sec)
Transport of proteins into nucleus
- Translation of protein finishes on free ribosomes in cytosol –> protein folds (signal sequence fully functional)
- Nuclear import receptor (VERY LARGE) binds to nuclear localization signal
- Protein and receptor enter nucleus –> then dissociate
Features of nuclear pore complexes
- Small molecules (even small proteins) freely pass through
- Passage of larger proteins is active (req. energy)
- Nuclear localization signal –> AA sequence tags protein for nuclear transport (import)
- Nuclear export signal tags protein for export
- Proteins pass through nuclear pore complexes W/OUT unfolding
What moves into the nucleus?
- Histones, proteins req. for transcription + DNA replication
- dNTPs, rNTPs
What moves out of the nucleus?
- Mature, properly processed mRNA
- Ribosomal RNA (manufactured in nucleolus)
Protein import into mitochondria
- Synthesis on free ribosomes in cytosol
- Signal sequence binds to import receptor on outer mito membrane
- Import receptor migrates to matching translocator in inner membrane
- Protein folding is undone and protein is fed through straight (very energy intensive)
- Localization sequence is cut off –> protein refolds in mito matrix
Transport across membranes (mito/chloro)
- Mito/chloro have double membranes
- Even though they have own genome + ribosomes –> most of their proteins encoded by nuclear genome –> must be imported
- Signal sequence located at N terminus of protein
- Proteins must be moved across both membranes at special sites where layers are in contact
- Subsequent transport within organelle req. another signal sequence (exposed after first one removed)
ER + endomembrane system
- ER is most extensive of endomembrane system
- Serves as entry point for proteins for ER + rest of endomembrane system (Golgi, lyso/endosomes), cell surface, secretory proteins
- Once in ER (in membrane/lumen) –> proteins NEVER re-enter cytosol
Transport into ER
- Synthesis BEGINS on free ribosomes in cytosol
- AS TRANSLATION OCCURS –> ER signal sequence recognized by SRP (they bind)
- SRP binds to SRP receptor in ER membrane –> brings growing polypeptide (+ ribosome) to translocation channel –> SRP displaced + recycled
- SRP receptor detaches + can go assist another protein transport
- Signal sequence forces translocation channel open + remains there since it is hydrophobic
- Ribosome still translating –> pushing peptides into ER
2 types of proteins transferred to ER
- Water soluble: translocated completed across into ER lumen (destined for lumen of organelle/secretion)
- Transmembrane proteins: translocated only partially across (destined for plasma membrane, ER membrane, membrane of another organelle) (not snipped off)
Water soluble proteins
Fully translocated into ER lumen, signal sequence cleaved off, folds inside
Single-pass transmembrane protein
- Have a hydrophobic stop-transfer sequence –> also gets stuck in translocation channel –> rest protein translated outside of ER
- Signal/start transfer sequence cleaved off
Multi-pass transmembrane protein
Multiple hydrophobic start/stop transfer sequences dictating which parts of protein on cytosolic vs non-cytosolic side
Temporary vesicles
- Allow material to leave + enter cells
- Move material b/w endomembrane compartments
- Carry soluble proteins (in their lumens) to plasma membrane for secretion
- Move membrane proteins (in their membranes) to be expressed on cell surface
- NOT considered organelles
Vesicle traffic
- Outward from ER (membrane added to plasma membrane): Golgi –> other organelles? plasma membrane?
- Inward (membrane subtracted from plasma membrane): plasma membrane –> endosomes (chemical changes happening) –> lysosomes
High specificity of destination
Vesicle budding
Protein coated pit forms –> membrane bent inward to form vesicle –> vesicle closed + plasma membrane sealed off
Clathrin coated vesicles
- Mediate transport from outward face to Golgi + inward from plasma membrane
- Forms basket that gives vesicle shape
- Adaptins capture specific cargo molecules by trapping receptors that bind to them
Cargo binds to receptors –> adaptin recognizes + bind receptor + clathrin –> vesicle forms + broken off by dynamin (req. energy) –> coated removed –> naked transport vesicle (can now be processed)
Vesicles finding their destination
- Must recognize + dock w/ its specific organelle
- Each transport vesicle displays molecular markers that identify its origin + cargo
- Markers must be recognized by complementary receptors on target membrane
Rab proteins
Family of monomeric GTPases, displayed on vesicle surface
Tethering proteins
- Displayed on cytosolic side of target membrane
- Docking –> interaction btw Rab + tethering protein
SNAREs
Transmembrane proteins on vesicle (v-SNARE) + target membrane (t-SNARE) –> consolidate docking + catalyze membrane fusion
Vesicle docking
Tethering: tethering protein (on membrane) recognizes Rab protein (on vesicle) + brings vesicle close to membrane
- Docking: If v-SNARE + t-SNARE match –> interact (specificity) –> pull vesicle closer to membrane on both sides –> lipid bilayers fuse
MEMBRANE ASYMMETRY MAINTAINED
ER processing
Most proteins covalently modified in ER
- Disulfide bonds
- Glycosylation (addition of sugar groups): various functions depending on protein, protect from degradation, help direct protein to proper organelle (act as transport signal for packing into appropriate vesicle), on cell surface –> cell-cell recognition
Disulfide bonds
- Covalent stabilization of protein structure found in secreted proteins (destined for more hostile extracellular environment)
- Formed in ER (oxidizing environment)
Glycosylation (ER)
- Addition of sugar groups during protein translocation into ER
- Various functions depending on protein: protect from degradation, help direct protein to proper organelle (act as transport signal for packing into appropriate vesicle), on cell surface –> cell-cell recognition
- Already formed carbohydrates attached to membrane lipid dolichol
- As growing polypeptide enters ER, carbohydrate group transfers to amino (NH2) groups of asparagine (Asn) side chains via membrane-bound enzyme (N-linked glycosylation)
IN ER LUMEN
Proteins leaving ER
- Only properly folded –> allowed to leave ER
- If misfolded –> chaperone protein surrounds it + gives another chance to fix
Unfolded protein response (UPR)
- Sensors for misfolded proteins activated in ER lumen –> transcription regulators activated (drive transcription of particular genes) –> activation of chaperone genes + other genes that increase protein folding capacity + expansion of ER
- If cell can’t keep up, UPR will trigger cell death –> apoptosis
Golgi apparatus structure
- Series of flattened sacs (cisternae)
- Organized into functionally distinct compartments w/ cis (entry) face closest to ER, trans (exit) face at other end
- Cis: newly formed
- Trans: breaking away
Functions of Golgi
Modification of new proteins arriving from ER (post-transcriptional modifs):
- Peptide chains shortened by proteases
- AAs modified
- CHO groups –> added in ER modified or removed
- Glycosylation: different CHO groups added to different AAs (O-linked glycosylation)
Most complex polysaccharide synthesis:
- Glycos amino glycans in extracellular matrix (animals)
- Pectins, hemicellulose (plant cell walls)
Regulated secretion
- Secretory proteins in high conc. in vesicles leaving Golgi
- Extracellular signal (hormone/neurotransmitter) binds to receptor –> secretion
- Cell is often “famous for secreting this particular protein” (main function)
Constitutive secretion
- Happening by default, all the time
- Vesicle buds off Golgi –> non-specific proteins/membrane lipids inside –> secretion
- Happening in all cells in an unregulated way
- e.g. Getting new lipids to plasma membrane, secreting products of cell (characteristic molecules –> cell conditioning its medium, creating comfort for itself)
Endocytic pathways
- Taking substances into cell by surrounding them w/ membrane –> become membrane bound vesicle
- 2 main types based on size: pinocytosis + phagocytosis
- 1 more type in animal cells: receptor-mediated endocystosis
Pinocytosis
- “Cell drinking” / “sampling”
- Tiny vesicles formed (endosomes)
- Done by all euks
- Solutes, macromolecules, fluid
- ‘Bulk’ –> any molecules present in enclosed fluid enter cell
- Non-specific + unregulated
Phagocytosis
- “Cell eating”
- Much larger vesicles (phagosomes)
- Done only by specialized cells –> phagocytes –> e.g. macrophages
- Particles, other cells, debris
Receptor-mediated endocytosis
- Very selective concentrating mechanisms
- Requires specialized receptors
- Particular molecules (ligands) for which the membrane has receptors
- Receptors grouped in patches of membrane called coated pits (e.g. clathrin)
LDL example of receptor-mediated endocytosis
LDL binds to receptors –> clathrin coated vesicle forms –> uncoats –> fusion with endosome –> contents transfer to lysososme (receptor buds off of transport vesicles + returns to plasma membrane)
Lysosomes
- Site of cellular digestion (from endocytosis, phagocytosis, and autophagy (break down of worn out mito/organelles)
- Have many acid hydrolases (work in acidic conditions)
- Have proton pump
- If mutation in acid hydrolase –> buildup of non-broken down material –> lysosomal storage diseases
Roadmap of protein traffic
- Escorted transport: cytosol –> nucleus
- Transmembrane transport: cytosol –> mitochondria/ER
- Vesicular transport: ER <–> Golgi –> endosomes, cell surface, secretory vesicles
- Endocytosis: cell surface –> early endosome –> late endosome –> lysosome
