Introduction and protein sorting - JU Medicine...Lipid rafts and diseases •HIV virus •Budding may occur from lipid rafts •Influenza virus •Raft-associated glycoproteins in

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

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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