Introduction and protein sorting - JU Medicine...Lipid rafts and diseases •HIV virus •Budding...
Transcript of Introduction and protein sorting - JU Medicine...Lipid rafts and diseases •HIV virus •Budding...
Introduction and protein sorting
Major components of cells
• Nucleic acids
• Carbohydrates
• Proteins
• Lipids (50% of mass of plasma membranes, 30% of mitochondrial membranes, 80% of myelin sheeth), species dependent
Me
mb
ran
e
pro
tein
s
Composition and properties of membranes
• Lipids: phospholipids, glycolipids, and cholesterol
• Cholesterol: animal, rigidity, fluidity
• It is not present in bacteria or plant cells
Composition and properties of plasma membranes
• Phospholipids are asymmetrically distributed between the two halves of the membrane bilayer
• The outer leaflet: choline, sphingomyelin
• The inner leaflet:ethanolamine, serine, inositol (minor)
• inositol has a role in cell signaling.
• serine stimulates apoptosis when extracellular.
Lipid rafts• Clusters of cholesterol and
sphingolipids (longer and straighter).
• Sphingolipids provide an ordered lipid environment.
• Rafts are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, as well as proteins involved in signal transduction and intracellular trafficking (endo- & exo-cytosis).
Lipid rafts and diseases• HIV virus
• Budding may occur from lipid rafts
• Influenza virus
• Raft-associated glycoproteins in envelope
• Prion disorder
• Normal prion protein (PrPc) is converted to abnormal proteins (PrPsc) in lipid rafts (aggregation).
Membrane proteins
• Peripheral: indirect, mainly ionic, pH or salt.
• Integral: transmembrane• α-helix: 20-25 a.a non-polar
• β-sheet: barrel
• Detergents
Lipid-anchored membrane proteins
• Myristoylation: N-terminus glycine
• Palmitoylation: sulfur of internal cysteine
• Prenylation: linking of "isoprene"-based groups
• Farnesylation: RAS (oncoprotein), 95% of pancreatic cancers
• sulfur of C-terminus cysteine
• Glycolipid (glycosyl phosphatidylinositol) anchors GPIs
• The carbohydrate bridges the protein with the fatty acid chains of the phospholipid (usually ethanolamine)
Protein mobility• Proteins (as lipids) are able to diffuse laterally
• Mobility restrictors:
• Cytoskeleton association
• Specific membrane domains such as tight junctions
• Lipid composition (e.g. lipid rafts)
Glycocalyx
• A carbohydrate coat
• Formed by oligosaccharides of glycolipids and transmembrane glycoproteins
• Functions:• Cell-cell interactions (e.g, leukocytes and selectins)
• Protection from ionic and mechanical stress
• Formation of a barrier for microorganisms
Endoplasmic reticulum (ER)• It is a network of membrane-enclosed tubules and sacs (cisternae) that extends from
the nuclear membrane throughout the cytoplasm
• It is the largest organelle of most eukaryotic cells
• Rough ER: covered by ribosomes
• Smooth ER: lipid metabolism, Ca++ stores
• Transitional ER: exit of vesicles to Golgi apparatus
• Microsomes
Protein sorting• Free ribosomes: cytosolic, nuclear,
peroxisomal, and mitochondrial proteins
• Membrane-bound ribosomes: others (most proteins) are transferred into the ER while they are being translated (cotranslationaltranslocation)• Stay there or sorted: golgi, peroxisomal
membrane, vesicles, plasma membrane, or secreted extracellularly
Ribosomal and protein targeting• All protein synthesis initiates on ribosomes that are free in the cytosol.
• Ribosomes are targeted for binding to the ER membrane by the amino acid sequence of the polypeptide at the amino terminus called a signal sequence (hydrophobic, ≈20, basic).
• It is then cleaved from the polypeptide chain during its transfer into the ER lumen, preproteins!.
Mechanism of translocation(co-traslational translocation)
• Step 1: recognition by the signal recognition particle (SRP), blockage
• Step 2: binding; SRP escorts the complex to the ER membrane (SRP receptor)
• Step 3: release; SRP is released, the ribosome binds to a translocon, and the signal sequence is inserted into a membrane channel
• Step 4: Translation resumes, and the growing polypeptide chain is translocated across the membrane
• Step 5: Cleavage of the signal sequence by signal peptidase releases the polypeptide into the lumen of the ER
Mechanism of translocation
Translocon
Posttranslational translocation
• Free ribosomes, remain unfolded (chaperones)
• Signal sequences recognized by a protein complex associated with the translocon
• The protein complex is also associated with a chaperone protein (BInding Protein - BiP), which drives protein translocation into the ER Translocon
Pathways of protein sorting• Lumen of ER or Golgi is similar to outside
• ER lumen: Secretory, ER, Golgi apparatus, and lysosomal proteins
• ER membrane: Membranous proteins
• Considerations
• Single vs. multiple membrane spanning region
• Orientation of N- and C-termini
Insertion of a membrane protein• A cleavable amino-terminal signal sequence that initiates translocation
across the membrane, and
• A transmembrane stop-transfer sequence that anchors the protein in the membrane
Stop transferClose channelMove laterally
Cleave signal sequence
Signal peptidase
Close channel
Insertion of a multi-domain membrane protein
Close channel
Re-openchannel
Once inside…Protein folding, assisted by the molecular chaperone, that keep protein unfolded until properly folded (e.g. BiP)
Disulfide bond formation by providing an oxidizing environment (the cytosol has a reducing environment) assisted by protein disulfide isomerase (PDI)
Assembly of multisubunit proteins
Also, once inside…
Specific glycosylation sequence Asn-X-Ser/Thr
Addition of glycolipid anchors to some plasma membrane
proteins. Carboxy-terminal
Functions of glycosylation:1.Prevents protein aggregation in the
ER2.Helps in further protein sorting
N-acetyl glucosamine is the first sugar
Fate of a glycoproteinER-associated degradation (ERAD)
• Calreticulin , a chaperone, releases it when glucose is removed
• A folding sensor binds to the protein
• If correctly folded, the protein moves to transitional ER
• If misfolded, glucose is added and calreticulin re-folds the proteins.
• If severely folded, the protein is degraded.
Unfolded protein response (UPR)
Activate UPR target genes such as
chaperones
BiP initiates
the process
Outcome:
1. General protein
synthesis
inhibition
2. ↑ Expression of
chaperones
3. ↑ Activity of
proteasomes
Protein sorting and retention• Many proteins with KDEL sequence (Lys-Asp-Glu-Leu) at C-terminus are retained
in the ER lumen
• If sequence is deleted, the protein is transported to the Golgi and secreted from the cell
• Addition of the sequence causes a protein to be retained in the ER
• The retention of some transmembrane proteins in the ER is dictated by short C-terminal KKXX sequences.
• Proteins bearing the KDEL and KKXX sequences appear to be recycled back to the ER
Protein sorting and retention• Membrane proteins contain di-acidic or di-Met signal sequences.
• They can also function as carriers of GPI-anchored and lumenal proteins.
Synthesis of phospholipids in ER
Enzymes (acyl transferase) are associated with the outer leaflet of
the membrane
Translocation of phospholipids across the ER membrane
Flippases
Synthesis of ceramide
Synthesis of other lipids• Steroid hormones are synthesized from cholesterol in ER
• Large amounts of smooth ER are found in steroid-producing cells, such as those in the testis and ovary
• Smooth ER is abundant in the liver, where it contains enzymes that metabolize various lipid-soluble compounds.
• The detoxifying enzymes inactivate a number of potentially harmful drugs (e.g., phenobarbital) by converting them to water-soluble compounds that can be eliminated from the body in the urine
ER-Golgi intermediate compartment (ERGIC)
Golgi apparatus and vesicular transport
• Functions of Golgi
• Further protein processing and modification (e.g. glycosylation)
• Protein sorting
• Synthesis of glycolipids and sphingomyelin
Structure of the Golgi
, endosomes
1. Cis Golgi network – protein entrance
2. Golgi stacks: medial & trans –most of the metabolic activities
3. Trans Golgi network – sorting and distribution center
Protein modification takes place at all levels
Processing of oligosaccharides in GolgiN-linked glycoproteins
Lysosomal vs. Membrane
Processing of oligosaccharides in GolgiO-linked glycoproteins
• Proteins can also be modified by the addition of carbohydrates to the side chains of acceptor serine and threonine residues.
• The serine or threonine is usually linked directly to N-acetylgalactosamine, to which other sugars can then be added.
Lipid and Polysaccharide Metabolism in the Golgi
• Transfer of phosphorylcholine group is from phosphatidylcholine to ceramide.
• Sphingomyelin is synthesized on the lumenalsurface.
• Addition of sugar residues.
• Glucose is added to ceramide on the cytosolicside and glucosylceramide then apparently flips and additional carbohydrates are added on the lumenal side of the membrane
Ceramide is synthesized in the ER
Protein Sorting and ExportIn contrast to the ER, all of the proteins retained within the Golgi complex are associated with the Golgi membrane rather than being soluble proteins within the lumen
Continuous, unregulated secretion
• Regulated secretion after signaling from specialized vesicles
• Protein packaging mediated by cargo receptor
• processing in Immature secretory vesicles
Transport to the plasma membrane of polarized cells
• This is accomplished by the selective packaging of proteins into transport vesicles from the trans Golgi or recycling endosomes
• Targeting is determined by special sequences (basolateral) or sugar modification (apical)
Processing of lumenal lysosomal proteins
Addition of N-acetylglucosamine
phosphates
Removal of N-acetylglucosamine
The enzyme recognizes a signal patch (a three-dimensional structural determinant) not a sequence
Transport of lysosomal proteins• Lumenal: marked by mannose-6-phosphates
bind to a mannose-6-phospahte receptor.
• Complexes are packaged into transport vesicles destined for late endosome, which mature into lysosomes.
• lysosomal membrane proteins are targeted by sequences in their cytoplasmic tails, rather than by mannose-6-phosphates.
Formation & fusion of a transport vesicle
Coat disassembly
Vesicular docking &
fusion
Vesicular transport
Coat proteins
Formation of clathrin-coated vesicles
Role of ARF1 (G-protein)(ADP-ribosylation factor)
1. Activation of Arf1 by GEF2. Recruitment of AP1 (not shown) and clathrin3. Formation of Arf1-clathrin-receptor-cargo complex4. Formation of vesicle5. Budding and transport of vesicle6. Inactivation of Arf1 and disassembly of coat7. Vesicle fusion
Players of vesicle fusion – SNARE & Rab• Rab: G-protein
• Function in several steps of vesicle trafficking
• SNARE: formation v-SNAREs-t-SNAREs complexes• Soluble NSF(N-ethylmaleimide-sensitive factor)
Attachment Protein) REceptor
• Effector proteins allow for specific interaction
The mechanism of fusion
Fusion
Closer vesicle-target
Disassembly of SNARE complex
Interaction of effector proteins
Tethering,hydrolysis of GTP, SNARE interactions
RECAP1. Interaction between different effector proteins
2. Tethering of the SNAREs
3. Hydrolysis of the GTP to GDP
4. The snares interact with each other
5. The vesicle membrane gets closer to the plasma membrane and then it dissolves by fusing into it
6. The SNARE complex is disassembled
7. The vesicle becomes part of the membrane
Exocytosis
Exocyst: A complex of 8 different proteins
Griscelli syndrome (GS) • A rare genetic condition
• Many Types: GS1, GS2, GS3
• Mutations in MYO5A, RAB27A and MLPH genes that encode the MyoVA-Rab27a-Mlph protein complex that function in melanosome transport & fusion
Griscelli syndrome (GS) • Pigmentary dilution of the skin, silver-grey hair, melanin clumps within hair
shafts
• Mature melanosomes accumulate in the centre of melanocytes