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Questions and Answers
What is the primary mechanism by which vesicles transport membrane and soluble proteins?
What is the primary mechanism by which vesicles transport membrane and soluble proteins?
What happens to proteins destined for the lysosome after leaving the trans-Golgi network?
What happens to proteins destined for the lysosome after leaving the trans-Golgi network?
Which types of vesicles are involved in the regulated secretion pathway?
Which types of vesicles are involved in the regulated secretion pathway?
What types of proteins are packaged into anterograde transport vesicles?
What types of proteins are packaged into anterograde transport vesicles?
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What process allows for the docking of vesicles to their target membranes?
What process allows for the docking of vesicles to their target membranes?
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Which of the following accurately describes vesicle budding?
Which of the following accurately describes vesicle budding?
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What is a characteristic feature of coated vesicles?
What is a characteristic feature of coated vesicles?
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Which of the following statements about secreted proteins is correct?
Which of the following statements about secreted proteins is correct?
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Which of the following accurately describes the direction of transport for COPII vesicles?
Which of the following accurately describes the direction of transport for COPII vesicles?
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What is the function of GTPase proteins in the assembly of protein coats for vesicles?
What is the function of GTPase proteins in the assembly of protein coats for vesicles?
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Which coated vesicle is primarily responsible for retrograde transport from the cis-Golgi back to the RER?
Which coated vesicle is primarily responsible for retrograde transport from the cis-Golgi back to the RER?
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How do vesicles select their cargo proteins for transport?
How do vesicles select their cargo proteins for transport?
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What triggers the dissociation of the protein coat during COPII vesicle synthesis?
What triggers the dissociation of the protein coat during COPII vesicle synthesis?
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Which protein is specifically involved in the polymerization of COPII coatings during vesicle budding?
Which protein is specifically involved in the polymerization of COPII coatings during vesicle budding?
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What is the role of Rab proteins in vesicle docking?
What is the role of Rab proteins in vesicle docking?
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Which of the following is true about Clathrin-coated vesicles?
Which of the following is true about Clathrin-coated vesicles?
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What is the role of Rab-GTP in vesicle docking?
What is the role of Rab-GTP in vesicle docking?
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Which proteins are involved in the SNARE complex on the vesicular and target membranes?
Which proteins are involved in the SNARE complex on the vesicular and target membranes?
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What triggers the dissociation of the SNARE complex after vesicle fusion?
What triggers the dissociation of the SNARE complex after vesicle fusion?
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What type of vesicular transport is associated with recycling membrane bilayers?
What type of vesicular transport is associated with recycling membrane bilayers?
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What is the primary function of the KDEL sequence in proteins?
What is the primary function of the KDEL sequence in proteins?
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How are COPII vesicles primarily involved in protein transport?
How are COPII vesicles primarily involved in protein transport?
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What occurs shortly after the release of a vesicle during membrane fusion?
What occurs shortly after the release of a vesicle during membrane fusion?
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Which statement accurately describes the structure of the Golgi apparatus?
Which statement accurately describes the structure of the Golgi apparatus?
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Which outcome does NOT align with the fate of proteins synthesized on bound ribosomes?
Which outcome does NOT align with the fate of proteins synthesized on bound ribosomes?
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What is the primary role of transport vesicles in the secretory pathway?
What is the primary role of transport vesicles in the secretory pathway?
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What occurs during cisternal progression in the Golgi apparatus?
What occurs during cisternal progression in the Golgi apparatus?
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Which type of secretion involves the storage of proteins inside the cell until a signaling event triggers release?
Which type of secretion involves the storage of proteins inside the cell until a signaling event triggers release?
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What distinguishes the role of v-SNAREs and t-SNAREs in vesicular transport?
What distinguishes the role of v-SNAREs and t-SNAREs in vesicular transport?
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Which type of vesicle is primarily involved in transporting proteins from the trans-Golgi to the lysosome?
Which type of vesicle is primarily involved in transporting proteins from the trans-Golgi to the lysosome?
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How is the cargo of proteins selected for transport within vesicles?
How is the cargo of proteins selected for transport within vesicles?
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What feature defines the trans-Golgi network (TGN) in the secretory pathway?
What feature defines the trans-Golgi network (TGN) in the secretory pathway?
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What initiates the assembly of COPII vesicle coats on the parent membrane?
What initiates the assembly of COPII vesicle coats on the parent membrane?
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Which feature is responsible for selecting specific cargo proteins during vesicle formation?
Which feature is responsible for selecting specific cargo proteins during vesicle formation?
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What leads to the recruitment of Sar1-GDP to the parent membrane?
What leads to the recruitment of Sar1-GDP to the parent membrane?
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Which statement accurately describes the role of Rab proteins in vesicle transport?
Which statement accurately describes the role of Rab proteins in vesicle transport?
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What is the function of the polymerized coat during vesicle budding?
What is the function of the polymerized coat during vesicle budding?
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Which GTP-binding protein is specifically associated with COPII vesicle synthesis?
Which GTP-binding protein is specifically associated with COPII vesicle synthesis?
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How does Sar1-GTP influence COPII vesicle coat formation?
How does Sar1-GTP influence COPII vesicle coat formation?
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What happens to the protein coat after vesicle budding in COPII transport?
What happens to the protein coat after vesicle budding in COPII transport?
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What occurs after the fusion of a vesicle with the target membrane?
What occurs after the fusion of a vesicle with the target membrane?
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Which component of the SNARE complex is a v-SNARE located on the vesicular membrane?
Which component of the SNARE complex is a v-SNARE located on the vesicular membrane?
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What is the primary role of the KDEL sequence in protein trafficking?
What is the primary role of the KDEL sequence in protein trafficking?
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What role does NSF play in the dissociation of the SNARE complex?
What role does NSF play in the dissociation of the SNARE complex?
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Which vesicle type is primarily responsible for mediating retrograde transport?
Which vesicle type is primarily responsible for mediating retrograde transport?
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What stabilizes the interaction between SNARE proteins during membrane fusion?
What stabilizes the interaction between SNARE proteins during membrane fusion?
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What happens to the coat of COPII vesicles shortly after their release?
What happens to the coat of COPII vesicles shortly after their release?
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What is the structure of the Golgi apparatus primarily characterized by?
What is the structure of the Golgi apparatus primarily characterized by?
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Study Notes
Protein Transport Mechanisms
- COPII - anterograde transport from the endoplasmic reticulum (ER) to the Golgi apparatus.
- COPI - retrograde transport between Golgi cisternae and from the cis-Golgi back to the ER.
- Clathrin-coated vesicles - transport between the plasma membrane or trans-Golgi to late endosomes.
Assembly of Protein Coats
- All vesicle coats contain a GTP-binding protein which regulates coat assembly.
- COPI and clathrin vesicles - contain ARF (ADP-ribosylation factor).
- COPII vesicles - contain Sar1.
- ARF and Sar1 - belong to the GTPase superfamily.
COPII Vesicle Synthesis
- Sar1-GDP is recruited by Sec12 on the parent membrane.
- Sec12 catalyzes the exchange of GDP for GTP on Sar1.
- GTP binding causes a conformational change in Sar1, exposing a hydrophobic N-terminal region.
- This region anchors the Sar1-GTP complex to the membrane and drives polymerization of COPII proteins, leading to coat formation.
COPII Vesicle Budding
- The coat forms and the vesicle buds from the parent membrane.
- Sar1 GTPase activity, with the help of COPII subunits, hydrolyzes GTP.
- This triggers protein coat dissociation, exposing integral proteins called v-SNAREs (vesicle-associated membrane proteins) on the vesicle membrane.
Targeting Cargo Proteins
- The vesicles select cargo proteins by binding sorting sequences present on the cargo proteins.
- These sorting sequences make molecular contacts with coat proteins.
- The vesicle coat selects cargo molecules by directly binding specific sorting signals on the cytosolic portion of membrane cargo proteins.
- The polymerized coat acts as an affinity matrix that clusters selected membrane cargo proteins into forming vesicle buds.
Docking of Vesicles to Target Membranes
- Requires a different set of GTP-binding proteins called Rab proteins (Rab GTPases).
- Cytosolic Rab-GDP is converted to Rab-GTP.
- This conformational change enables the Rab-GTP complex to anchor to the vesicle membrane.
Secretory Pathway
- Initially studied in secretory cells that produce large quantities of proteins like insulin and digestive enzymes.
Fates of Proteins Synthesized on Bound Ribosomes
- Proteins made that follow the secretory pathways but are not meant to stay in the ER or Golgi have the following fates:
- Regulated Secretion - proteins are stored in secretory vesicles and released upon a specific signal.
- Constitutive Secretion - proteins are continuously released from the cell.
- Directed to the Lysosome - proteins are targeted to the lysosome for degradation.
- Insertion into the Plasma Membrane - proteins are incorporated into the plasma membrane.
Principle of Secretion
- Transport of membrane and soluble proteins from one membrane-bound compartment to another is mediated by transport vesicles.
- Vesicles collect cargo proteins in buds arising from the membrane of one compartment and deliver the cargo to the next compartment by fusing with the membrane of that compartment.
- At least three different types of vesicles are required for transport.
Secretory and Endocytic Pathways
- Newly made proteins are incorporated into the ER lumen or membrane and packaged into anterograde transport vesicles (forward-moving).
- Vesicles fuse with each other to form the cis-Golgi cisterna (flattened membrane-bound compartment).
- Some proteins are retrieved back to the ER via retrograde transport vesicles (backward-moving).
- New cis-Golgi cisterna with cargo move cis to medial to trans position.
- Retrograde transport of enzymes and Golgi-resident proteins needed at each position is constantly retrieved.
- Cargo reaches the trans-Golgi network (TGN) which is the major branch point where cargo is loaded into one of three vesicle types:
- Exocytosis - continuous secretion.
- Secretory vesicles - regulated secretion; stored inside the cell until a signal triggers exocytosis.
- Transport vesicle to lysosome via a late endosome (proteins can also enter this way via endocytosis).
Visualized Protein Transport
- Vesicular Stomatitis Virus (VSV) membrane glycoprotein fused to Green Fluorescent Protein (GFP) can be used to visualize protein transport.
Molecular Mechanism of Vesicle Budding and Fusion
- Vesicles move from parent/donor organelle to daughter organelle.
- Donor organelle contains integral membrane proteins called v-SNAREs that join with their cognate t-SNAREs (target-SNAREs) in the target membrane.
Types of Coated Vesicles
- COPII - anterograde transport from the ER to the Golgi.
- COPI - retrograde transport between Golgi cisternae and from the cis-Golgi back to the ER.
- Clathrin - transport between the plasma membrane or trans-Golgi to late endosomes.
Docking of Vesicles to Target Membranes
- Rab-GTP complex binds to a Rab effector on the target membrane.
- This allows for the fusion of the membrane and formation of SNARE complexes.
- VAMP (v-SNARE on the vesicle membrane) interacts with syntaxin and SNAP-25 (t-SNAREs on the target membrane).
- Multiple non-covalent interactions stabilize this structure.
Vesicle Fusion and SNARE Complex Dissociation
- After fusion, the Rab-GTP is hydrolyzed and released.
- The SNARE complex dissociates requiring ATP hydrolysis and other cytosolic proteins.
- NSF (N-ethylmaleimide-sensitive factor) associates with the SNARE complex with the aid of α-SNAP.
- NSF then hydrolyzes ATP, releasing sufficient energy to dissociate the SNARE complex.
Vesicle-mediated Protein Trafficking between the ER and cis-Golgi
- Anterograde transport - mediated by COPII vesicles, moves proteins to the Golgi.
- Retrograde transport - mediated by COPI vesicles, recycles membrane bilayer and some proteins.
- Shortly after vesicle release, the coat is shed, exposing proteins required for fusion.
Role of KDEL and KDEL Receptor
- KDEL (Lys-Asp-Glu-Leu sequence) is a retrieval signal for ER-resident luminal proteins from the Golgi.
- This retrieval system prevents depletion of ER luminal proteins, which are needed for proper folding of newly made secretory proteins.
Golgi Apparatus
- Composed of membrane-bound sacs known as cisternae.
- Typically, there are between 5-8 cisternae present.
Secretory Pathway
- The secretory pathway was first studied in cells that produce and secrete large quantities of proteins.
- Some examples of these proteins are insulin and digestive enzymes.
Fates of Proteins Synthesized on Bound Ribosomes
- Proteins synthesized on bound ribosomes that follow the secretory pathway have the following fates:
- Regulated Secretion
- Constitutive Secretion
- Directed to the Lysosome
- Insertion into the Plasma Membrane
- Proteins that are delivered to the plasma membrane include cell-surface receptors, transporters for nutrient uptake, and ion channels.
- Secreted proteins include digestive enzymes, peptide hormones, serum proteins, and collagen.
Principle of Secretion
- The transport of membrane and soluble proteins from one membrane-bound compartment to another is mediated by transport vesicles.
- Vesicles collect cargo proteins in buds arising from the membrane of one compartment and deliver the cargo by fusing with the membrane of the next compartment.
- There are at least three different types of vesicles required for transport.
Secretory & Endocytic Pathways that Sort Proteins
- Newly made proteins are incorporated into the ER lumen or membrane and packaged into anterograde transport vesicles.
- These vesicles fuse with each other to form cis-Golgi cisterna.
- Some proteins are retrieved back to the ER via retrograde transport vesicles.
Secretory & Endocytic Pathways that Sort Proteins
- New cis-Golgi cisterna containing cargo move from the cis to medial to trans position.
- Retrograde transport of enzymes and Golgi-resident proteins is needed at each position and constantly retrieved.
- When the cargo reaches the trans-Golgi network (TGN) it is loaded into one of three vesicles:
- Exocytosis: continuous secretion
- Secretory vesicles: regulated secretion
- Transport vesicle to the lysosome via a late endosome.
Vesicular Stomatitis Virus (VSV) membrane glycoprotein fused to Green Fluorescent Protein (GFP)
- VSV membrane glycoprotein fused to Green Fluorescent Protein (GFP) can be used to visualize protein transport.
Molecular Mechanism of Vesicle Budding and Fusion
- Vesicles move from the parent/donor organelle to the daughter organelle.
- The donor organelle contains integral membrane proteins called v-SNAREs that join with their cognate t-SNAREs in the target membrane.
3 Types of Coated Vesicles
- Each type of coated vesicle has a different protein coat.
- Each is formed by reversible polymerization of distinct protein subunits.
- The different types of coated vesicles are:
- COPII: transports in the anterograde direction from the RER to the Golgi.
- COPI: transports in the retrograde direction between Golgi cisternae and from the cis-Golgi back to the RER.
- Clathrin coated vesicles: transports between the plasma membrane or trans-Golgi to late endosomes.
Assembly of Protein Coats
- A conserved set of GTPase switch proteins control the assembly of different vesicle coats.
- All vesicles contain a GTP-binding protein that regulates assembly.
- COPI and Clathrin vesicles contain ARF (ADP-ribosylation factor).
- COPII contains Sar1.
- ARF and Sar1 belong to the GTPase superfamily.
COPII Vesicle Synthesis
- Sar1-GDP is recruited by Sec12 on the parent membrane.
- Sec12 catalyzes the reaction that exchanges GDP for GTP binding.
- The binding of GTP causes a conformational change in Sar1.
- This conformational change exposes a hydrophobic N-terminal region that tethers the Sar1-GTP complex to the membrane and drives polymerization of COPII proteins and coat formation.
COPII Vesicle Synthesis
- The coat forms and the vesicle buds from the parent membrane.
- Sar1 GTPase activity, with the help of some COPII subunits, hydrolyzes GTP which triggers protein coat dissociation.
Targeting Cargo Proteins
- Vesicles select cargo proteins by binding sorting sequences present on cargo proteins.
- Cargo targeting sequences make specific molecular contacts with coat proteins.
- The vesicle coat selects cargo molecules by directly binding specific sorting signals on the cytosolic portion of membrane cargo proteins.
- The polymerized coat is an affinity matrix that clusters selected membrane cargo proteins into forming vesicle buds.
Docking of Vesicles to Target Membranes
- Docking requires another set of GTP-binding proteins known as Rab proteins (Rab GTPases).
- Cytosolic Rab-GDP is converted to Rab-GTP which causes a conformational change that enables the Rab-GTP complex to anchor to the vesicle membrane.
- The Rab-GTP complex binds to a Rab effector on the target membrane.
Docking of Vesicles to Target Membranes
- This allows for the fusion of the membrane and formation of SNARE complexes.
- VAMP = v-SNARE on the vesicular membrane.
- Syntaxin & SNAP-25 = t-snare on the target membrane.
- Multiple non-covalent interactions stabilize this structure.
Docking of Vesicles to Target Membranes
- After fusion, the Rab-GTP is hydrolyzed and released.
- The SNARE complex dissociates: requires ATP hydrolysis and other cytosolic proteins.
- NSF is an N-ethylmaleimide sensitive factor.
- NSF associates with the SNARE complex with the aid of α-SNAP.
- NSF then hydrolyzes ATP, releasing sufficient energy to dissociate the SNARE complex.
Vesicle-mediated Protein Trafficking between the ER & cis-Golgi
- Anterograde transport is mediated by COPII vesicles.
- Retrograde transport is mediated by COPI vesicles.
- Anterograde transport moves proteins to the Golgi.
- Retrograde transport recycles membrane bilayer and some proteins.
- Shortly after the vesicle release, the coat is shed exposing proteins required for fusion.
Role of KDEL & KDEL Receptor
- KDEL - Lys-Asp-Glu-Leu sequence.
- Usually found as the C-terminal sequence.
- This retrieval system prevents the depletion of ER luminal proteins like those needed for proper folding of newly made secretory proteins.
Golgi Apparatus
- The Golgi apparatus is composed of membrane-bound sacs called cisternae, usually between 5-8 in number.
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Description
Test your understanding of protein transport mechanisms, including COPII and COPI pathways. This quiz covers details about vesicle coats, GTP-binding proteins, and the assembly process essential for intracellular transport. Enhance your knowledge about the role of ARF and Sar1 in vesicle synthesis.
