Protein Transport Between Organelles

Pathway of Proteins into the ER

1. Polypep is synthesized until ER signal sequence is formed. Then SRP binds to signal sequence and blocks further translatoin

2. SRP binds to the SRP receptor which is close to the translocon. SRP is released and polypep is inserted into translocon

   1. SRP is released by GTP hydrolysis which reduces affinity

3. ER sig. sequence contacts the interior of the translocon which displaces the plug and opens the channel to the ER.

   1. Signal peptidase cleaves off signal sequence

   2. Chaperons binds to facilitate folding

4. When finished, polypep is released to lumen and ribosome detaches from membrane, subunits dissociate and release mRNA

Signal for Protein going to ER

15-30 a.a sequence, 6-12 core hydrophobic a.a at the N term. It is proceeded by a hydrophilic region

Receptor for Protein going to ER and Nature of Translocation Channel

SRP receptor binds to SRP, it is an integral membrane protein.

Translocon. Gated channel

Source of Energy for Proteins going into the Er

GTP hydrolysis to release SRP from SRP receptor

Protein Transport from ER to cis-golgi

SAR1-GDP is recruited to the donor membrane where GEF Sec 12 replaces GDP with GTP to cause the N-terminal alpha helix in Sar 1 to be accessible and stick in the membrane

SAR1-GTP recruits Sec 23 and Sec 24 to cause a curve conformation and bending of membrane

Sec 24 is also an adaptor protein to bind with the cargo receptor to recruit proteins to the forming vesicle

Integral membranes are targeted to the vesicle like v-snares which is needed for fusion to correct target membrane

Sec13 and Sec 31 form the cage-like outer layer. It is a simple lattice with each vertex a convergence of 4 sec 13-sec31 dimers

SAR1-GTP is hydrolyze to SAR1-GDP to disassemble the protein coat and allows the v-SNARES to target the proper membrane

Direction of Transport and type of Coat Protein for Er to cis golgi transport

Anterograde and COP2

Pathway for Protein going from TGN to Lysosome

• When cis golgi matures to trans, and encounters mannose 6-phosphate receptor the protein will bind to it. (anything with mannose 6-phosphate will bid)

• The receptor will bind to GGA adaptor that is also associated with Arf1-GTP and clathrin

• At low pH the affinity between MPR and enzyme is low and is able to release at the lysosome because of its low pH

  • Endosomes have a V pump which decrease internal pH by pumping protons to create the lysosome

Signal for proteins going from TGN to Lysosome and Receptor

Signal/tag is a mannose 6-phosphate that’s added at the cis-golgi

The lysosomal enzyme is created at the ER and glycosylated then the enzymes, then at the cis-golgi it addes phosphate on the 6th carbon in mannose

Mannose 6-phosphate receptor

Source of Energy for proteins going from TGN and Lysosome

GTP hydrolysis to remove clathrin coat

Pathway for Exocytosis: Constitutive Secretion

After budding from TGN vesicle will move directly to cell surface and immediately fuse, it is continuous/If there is no targeting sequence and it's form the golgi, it will be secreted out or join as membrane protein

Pathway for Exocytosis: Regulated Secretion

Vesicles will stay near the membrane and only fuse in response to specific signals (like insulin).

Fusion is triggered by hormonal or chemical signals

 

Polarize Secretion is when proteins are secreted from a limited region of the plasma membrane like Neurotransmitter at nerve junctions or digestive enzymes at the intestinal side of the cell

• Proteins and lipids are sorted into vesicles that have receptors that bind localized sites on the plasma membrane

Pathway for Bulk Phase Endocytosis

Clathrin-independent endocytosis

• Cell maintains membrane and fluid balance

  • Compensate for plasma membrane gain by exocytosis and maintains surface to volume ratio

  • Doesn't ingest particular molecules/non-specific

Pathway for Receptor-Mediated Endocytosis

Clathrin-dependent Endocytosis

• Ligand binds to its receptor on the outer cell surface

• Complex diffuse laterally until it encounters a coated pit, a site for collection and internalization of the complex

• Accumulation of complexes in the pit triggers the accumulation of clathrin-coat proteins (adaptor protein, clathrin and dynamin) on the cytosolic side of the membrane

  • It induces curvature and invagination

• Pit will pinch off and from a coated vesicle

How does the Clathrin Vesicle Form in Receptor Mediated Endocytosis

Clathrin molecules have 3 heavy chains and 3 light chains joined at the centre to make a triskelion

 

AP2 (Adaptor Protein 2) promotes the assembly of clathrin cage and recruitment of membrane receptors to budding vesicle.

• Binds with clathrin and endocytic cargo

In membrane, Phosphoinositide (PI[4,5]P2) binding will change the conformation of AP2 and makes the cargo binding site accessibly

As clathrin accumulates, dynamin will associate at junction forming a ring between the budding vesicle and plasma membrane

It is a cytosolic GTPase which when GTP hydrolyzes, dynamin rings tightens and eventually separating the vesicle from plasma membrane

Dynamin Mutation

• Unable to pinch off vesicle

• Junction continues to elongate

Receptor-Mediated Endocytosis of LDL

Sorting Signal

• On LDL receptor on cell membrane

• 4 amino acid motif on C-terminus of receptor NPXY (Asn-Pro-Any-Tyr) binds to ApoB

 

Pathway

• Cell surface LDL recep bind to ApoB of LDL by interaction between sorting signal of cytosolic domain of receptor and AP2 complex which incorpotates the receptor-ligand complex in vesicle

• Clathrin coated pits are pinched off by dynamin

• Clathrin coat is shed and vesicles fuse with late endosomes

• Late endosomes fuse with lysosomes and protein/lipids and broken down

• Receptor is recycled back to the surface

Structure of LDL

• Amiphatic shell

• Monolayer phospholipid

• Cholesterol

• 1 large protein ApoB

• Apolar core with cholesterol esters and triglycerides

What causes the released of receptor-ligand complex in late endosomes (Late endosomes)

• In endosomes histidine residue in the Beta-propeller domain of LDL receptors become protonated which then has a high affinity to the binding arm that has negatively charge residues

• This causes release of LDL particle

Phagocytosis

Contact with the target triggers the onset of phagocytosis

• Folds in the membrane, pseudopods,  surround the object to form an intracellular phagocytic vacuole (phagosome)

• Endocytic vesicles fuse with endosomes.lysosomes to degrade the target

Functions of Peroxisomes

• H2O2 metabolism

  • Catalase that degrade H2O2 into water and oxygen

  • Oxidate that oxidize organic substance and produce H2O2

• Detoxify harmful compounds

• Oxidation of fatty acids

• Metabolism of nitrogen-containing compouns

• Catabolism of unusual substances

Proteins are synthesize on free cytosolic ribosomes then imported post-translationally

Pathway for Peroxisomal Proteins

1. Proteins for the peroxisomal matrix binds the cytosolic Pex 5 receptor (makes Dimer with another Pex 5)

2. Interacts with Pex 14 at the membrane

3. Protein dissociate from Pex5 and translocate to matrix

4. Pec 5 is returned to cytosol by being ubiquitnylated by membrane proteins and then is removed by ATP hydrolysis by AAA-ATPase proteins

Signal for Peroxisomal Proteins

SKL on the C-terminal (serine, lysine, leucine)

 

• Folded protein is importedted into peroxisomes

• Signal is not cleaved

Energy Usage for Peroxisomal Protein Import

ATP hydrolysis is use to release Pec5 from membrane

Proteins that are imported to Nucleus

• Histones

• DNA and RNA polymerases

• Transcription factors

• RNA-processing proteins

• Ribosomal proteins

Pathway for Nuclear IMport

1. A cytoplasmic protein with NLS is recognized by importin and binds at NLs

2. Importin-protein complex docks at NPC and is transported into nucleus

3. RAN-GTP associates importin which causes release of cargo protein

4. Ran-GTP-importin complex is transported back to cytoplasm through NPC

5. GTP is hydrolyzed by GAP to release importin

6. Ran-GDP travels back into nucleus and encounters GEF to regenerate Ran-GTP

Signals for Nuclear Import

NLS, nuclear localization signal

• 8-30 a.a long has proline and positively charged amino acids like arginine and lysine

 

• Will import folded protein

• Signal not cleaved

Channel/Receptor used for Nuclear Import

• Specialized channels

• In the nucleoporins are FG-repeat domains (phenylalanine and glycine)

• In direct contact between cytosol and nucleoplasm

• Able to transport 1000 macromolecules a second in both direction

Energy Requirement for Nuclear Import

Energy is needed to transport large protein and RNA

Concentration gradients

• Ran GTP is low in cytosol and high in nucleus

• Ran GDP is high in cytosol and lowin nucleus

• Importin is high in the cytosol and low in the nucleus

 

Ran-GTP leaves the nucleus by flowing down its concentration gradient

 

GTP hydrolysis

• Hydrolysis causes release of importin in the cytosol to bind to new cargo protein

Proteins that must be exported from Nucleus

• tRNA

• mRNA

• RNA complexes

• Ribosomal subunits

Pathway of Nuclear Export

1. Ran-GTP binds to exportin and cargo proteins with NES in nucleus

2. Export occurs

3. GTP is hydrolyzed and cargo is released

4. Exportin is imported back into the nucleus

   1. Exportin must have NLS and NES

Signals for Nuclear Expore

NES, nuclear export sequences

• Short and leucine rich

• Lxx,Lxx,Lxx

 

• Import folded protein

• Signal not cleaved

Energy Requirement for Nuclear Export

Concentration Gradients

GTP hydrolysis

Mitochondrial Matrix Protein Import Pathway

1. Cytosolic proteins are boudn to chaperones to remain unfold

2. Presequence binds to the receptor component of TOM and is positioned into the channel component of TOM of outer membrane

3. During transport for matrix proteins, TOM and TIM23 are brought together

4. Movement into the matrix is powered by electrical potential as the matrix is more negative and the presequence is positive

5. 2 hypotheses as to how it's pulled in

   1. Chaperones use ATP hydrolysis to pull on peptide to bring it in

   2. Random diffusion allows peptide to poke the N-terminus through the channel and chaperones bin to prevent it from leaving. This occurs continuously

6. The presequence is cleaved by a peptidase and protein folds into its native conformation

SIgnal for Mitochondrial Matrix Protein

Presequence: positive and hydrophobic a.a residues on N-terminal as alpha helix

 

The presequence is cleaved by a peptidase and protein folds into its native conformation

Overall protein must be unfolded in order to fit into the channle

Receptor/translocase and nature of it for Mitochondrial Matrix Protein

TOM

• Translocase of the outher membrane

  • TOM has a receptor component and a channel

Channel is small so protein must be unfolded to fit

Energy Requirement for Mitochondrial Matrix Protein Import

ATP hydrolysis to release cytosolic chaperone after inserting the pre-sequence into the Tom complex and then the Tim complex

 

Membrane potential across the inner membrane powers the movement of the pre-sequence and polypep chain into the matrix

 

Mitochondrial chaperone (hsp70) bins to polypep chain in the matrix is ATP dependent

Inner Mitochondrial Membrane Protein Import Pathway

1. Cytosolic proteins are bound to chaperones to remain unfold

2. Presequence binds to the receptor component of TOM and is positioned into the channel component of TOM of outer membrane

3. For Inner membrane proteins, TOM and TIM 22 are brought together

4. The Inner targetting sequence signals TIM 22 channel to allow protein to pass into the lipid membrane

 

The presequence is cleaved by a peptidase and protein folds into its native conformation

Signal for Mitochondrial Inner Membrane Protein

Presequence: positive and hydrophobic a.a residues on N-terminal as alpha helix

Inner Targeting sequence: hydrophobic amino acids

Receptor/translocase and nature of it for Mitochondrial Inner Membrane Protein

TOM

• Translocase of the outher membrane

  • has a receptor and a channel component

TIM22

• Translocase of the inner membrane, into the matrix

  • allows protein to pass through the bilayer

 

Channel is small so protein must be unfolded to fit

Energy Requirement for Mitochondrial Inner membrane import

ATP hydrolysis to release cytosolic chaperone after inserting the pre-sequence into the Tom complex and then the Tim complex

Chloroplast Protein import

Stoma Protein

• Cytosolic chaperones bind to unfolded protein

• Enter TOC and TIC complex, Hsp 70 helps to pull it in and binds to hsp60 to fold

 Thylakoid proteins

• Must have a thylakoid transfer domain to enter it

All proteins have a stroma targeting domain (same as intermembrane space)

A thylakoid transfer domain is needed to enter the thylakoid

Stoma targeting domain is cleaved

TOC and TIC complex

ATP hydrolysis to release cytosolic chaperones

Targeting/Tethering Vesicle to Target

Direction of transport: Anterograde/Retrograde

Both in COP2 and COP1 vesicles

Pathway

Initial contact between vesicle and target membrane involves tethering proteins that are either rod-shaped fibrous proteins that form long bridges or multi-protein complex to keep the two membrane close

Membrane vesicle/target specificity is done by Rab GTPases family where a different Rab protein associates with different membranes in GTP bound form

• It recruits cytosolic tether proteins to membrane surface

Membrane Fusion

Uses v-SNARES/t-SNARES

• Mediates fusion

• Sorting and targeting of vesicles involves 2 families of SNARE (SNAP receptor)

 

V-SNARE are found on vesicles

t-SNARE are fuond on target membranes

• They are complimentary molecules and allow recognition between vesicles and its target

Pathway

• v- and t-SNARE alpha helices tightly interwine forming a 4 helical bundle and pull the membranes together

  • In vitro the interaction is strong enough to fuse

  • In vivo it requires a calcium concentration rise to initiate fusion

Once fused they are still tightly associates are in the same membrane

NSF and SNAPs is required to pry apart the SNARES using ATP hydrolysis to dissociate

Retrograde Transport GOlgi to ER/Golgi or TGN to CGN

• Preventing protein from leaving ER (retenttion)

• Returning others from the Golgi (retrieval)

1. Soluble ER-Specific Porteins

   1. Most common retrieval/retention tag is the KDEL. C terminal amino acid sequence (lys-Asp-Glu-Leu) mediate return of soluble proteins to ER

2. Er-Specific Transmembrane protein

   1. Membrane proteins have retrieval/retention tags on their cytosolic domain that bind to vesicle coat proteins to return to ER

   2. KKXX (lys-lys-any-any) is th emost common

   3. Other membrane compartment have different signal

General Pathway for Retrograde Vesicle Transport

Arf1-GDP uses Gea(a GEF) to exchange for GTP.

Arf1-GTP will then associate with the cargo and the COP1 complex (made of 7 proteins called the coatamer)

When enough COP1 complexs are present the vesicle will initiate budding (similar to COP2)

Retrograde transport of soluble ER resident proteins

• KDEL proteins are er resident proteins, when they escape they will associate with KDEL receptors that have efficient binding at lower pH environments like the golgi

• KDEL receptors mediate transport of KDEL proteins in Er-bound transport vesicles where KDEL receptors released the protein at a higher pH environment like the ER