exam 3.4 Protein Targeting II - Vesicle Based

Protein Targeting II - Vesicle Based
Overview of Protein Targeting Pathways

  • There are two primary protein targeting pathways:
      - Signal-based targeting
      - Vesicle-based targeting:
        - Involves the transport of proteins from the rough endoplasmic reticulum (ER) to their final destinations in the endomembrane system or for secretion.
        - Known as the secretory pathway.

Vesicle Based Targeting

  • Proteins are initially transported via signal-based targeting to the ER.

  • Upon reaching the ER:
      - Fate of proteins:
        - Some proteins remain in the ER.
        - Other proteins are modified and/or targeted to other locations such as:
          - Golgi apparatus
          - Nuclear envelope
          - Lysosomes/endosomes
          - Vacuoles
          - Plasma membrane
          - Outside of the cell

Vesicles and Topology

  • Vesicle:
      - Defined as small membrane-bound sacs that transport proteins between compartments of the endomembrane system through processes of budding and fusion.

  • Lumen:
      - Refers to the inner chamber of the vesicle.
      - The lumen of vesicles is topologically equivalent to the outside of the cell, indicating that the inside environment of the vesicle parallels extracellular conditions.

  • Topology:
      - Describes the orientation of molecules in membranes.

  • Proteins in the ER targeted elsewhere are transported via vesicles.

Overview of the Secretory Pathway

  • Proteins with a signal sequence (ss) are targeted to the ER.

  • Vesicle Transport Steps:
      - Vesicles bud off from the ER and migrate to the cis Golgi.
      - Involves processes of cisternal maturation within the Golgi apparatus.
      - From the trans Golgi network, proteins are sorted into different types of vesicles for trafficking to various destinations (e.g., primarily to the plasma membrane (PM) or lysosome).
      - Also encompasses processes such as retrograde transport from the Golgi back to the ER and endocytosis which employ similar mechanisms.

  • Importantly, proteins are never found in the cytosol during these transport processes.

Transport of Cargo Using Vesicles

  • Cargo:
      - Refers to the protein being transported.

  • Mechanism of Transport:
      - Receptors in the ER membrane bind to the protein leading to clustering of proteins aided by G-protein rab and SNARE proteins.
      - This process recruits coat proteins which facilitate the budding process.

  • Budding Process:
      - Coat proteins cluster around the membrane, inducing curvature which leads to vesicle formation.
      - After the vesicle buds off, GTP hydrolysis triggers coat disassembly, exposing v-SNAREs which are critical for target recognition.

Types of Coat Proteins

  • Specific coat proteins are associated with various types of vesicles, each identified for the specific transport processes they mediate:
      1. Clathrin-coated vesicles:
         - Mainly involved in exocytosis and endocytosis.
         - Function in transport between the plasma membrane and endosomes, the Golgi and plasma membrane, as well as Golgi-to-endosome transport.
      2. COPI-coated vesicles:
         - Primarily facilitate exocytosis or flow within the endomembrane system (EMS).
         - Transport between Golgi and plasma membrane, laterally between cis and trans Golgi, and retrieval from the ER to the Golgi.
      3. COPII-coated vesicles:
         - Specifically mediate transport from the ER to the cis side of the Golgi apparatus.

Close-Up on Coat Proteins

  • Coat proteins form clustered lattices by interacting with transmembrane proteins, effectively bending the membrane.
      - This membrane-bending ability is posited to have contributed significantly to the formation of the endomembrane system and the nuclear envelope (as suggested by Kovtun et al., 2020 and Dokudovskaya et al., 2005).

Close-Up on SNAREs

  • v-SNAREs (such as VAMP) and t-SNAREs (like syntaxin and SNAP25) are key proteins that mediate membrane fusion through their interactions.

  • The close approximation of the two membranes results in membrane fusion, which allows the cargo protein to transition into the Golgi apparatus.

  • The stability of SNARE complexes necessitates additional proteins (e.g., NSF and SNAP) to facilitate their separation and recycling, with ATP hydrolysis providing the required energy.

Overview of the Golgi Apparatus

  • The Golgi apparatus is structured as 4-6 stacks of membrane-bound compartments known as cisternae.

  • It exhibits directionality:
      - Materials enter from the cis side and exit from the trans side.

  • O-linked glycosylation may occur in this organelle.

  • Some proteins contain a Golgi-retention signal and are retained within the Golgi, while others are sorted for further transport.

Transport Through the Golgi

  • There are two models for transport through the Golgi:
      - Vesicular Shuttle Model:
        - Proposes that vesicles bud off and transport materials from one Golgi compartment to another.
      - Cisternae Maturation Model:
        - Suggests that the Golgi cisternae mature and migrate through the Golgi stack, carrying materials along with them.

Transport from the Golgi

  • Vesicles bud off from the trans-Golgi network, directing proteins to various destinations.

  • The type of coat proteins involved depends on the target location of the protein.

  • Recognition between protein sequences and their respective coat proteins along with G-proteins is essential for accurate vesicle assembly and targeting.

Transport from the Golgi to the Plasma Membrane

  • There are two primary pathways for the delivery of secreted proteins and membrane-bound proteins/lipids to the plasma membrane:
      1. Constitutive Secretory Pathway:
         - Operates continuously, with vesicles from the Golgi permanently fusing with the plasma membrane without requiring a sorting signal (default pathway).
      2. Regulated Secretory Pathway:
         - Active under specific conditions, vesicles form but do not fuse until a signaling event occurs (e.g., influx of Ca2+).
         - Common in processes such as neurotransmitter release and the secretion of digestive enzymes.

Regulated Secretory Pathway in Neurotransmitter Release

  • In neurotransmitter release, the regulated secretory pathway is employed.

  • Vesicles form but remain inactive until an influx of Ca2+, which occurs through voltage-gated Ca2+ channels activated by action potentials.

  • Ca2+ sensor proteins (like synaptotagmin) then interact with vesicle phospholipids or SNAREs to facilitate the fusion of vesicle membranes with plasma membranes.

Transport from the Golgi to Lysosomes

  • Multiple pathways direct vesicles toward lysosomes, including trafficking through late endosomes.

  • Key concepts regarding lysosomes:
      - Contain hydrolases that digest macromolecules and organelles.
      - Enzymes are synthesized as zymogens (inactive precursors) that become active post-cleavage.
      - Lysosomal pH is acidic (approximately 4.5-5.0), which is maintained by H+ pumps in the membrane, optimizing conditions for hydrolase functions.
      - The membrane is protected by heavily glycosylated proteins facing the lumen.

Formation of a Lysosome

  • The formation of a lysosome occurs through several stages:
      1. An early endosome emerges when the cell endocytoses material, forming a vesicle.
      2. This progresses to a late endosome as vesicles from the Golgi deliver hydrolytic enzymes.   3. Further maturation occurs with proton pumps reducing the pH, resulting in the transition from an endolysosome to a lysosome.

Golgi Traffic to Lysosomes

  • Hydrolases aimed at lysosomes possess a lysosomal-targeting signal recognized within the Golgi apparatus.

  • M6P (mannose six-phosphate) is introduced via N-linked glycosylation.

  • M6P is then recognized by its receptor, a transmembrane protein in the Golgi, which results in the creation of a lysosome-directed vesicle that fuses with late endosomes.

  • The acidic environment causes the dissociation of M6P from its receptor, allowing acid phosphatase to cleave off the phosphate from mannose, thus ensuring retention within the lysosome.

Extracellular Vesicles

  • Exocytosis of vesicles can also occur from the endosomal compartment, serving functions such as signaling or waste removal.

Endocytosis

  • Small vesicular buds can incorporate small molecules or droplets into cells, requiring energy for this process.

  • These vesicles can subsequently fuse with Golgi enzymes to form lysosomes, which digest the internalized substances.

Receptor-Mediated Endocytosis

  • Receptor-mediated endocytosis utilizes clathrin-coated pits to form vesicles that selectively carry specific substances into the cell.
      - This serves as a mechanism to deactivate signaling pathways by internalizing activated receptors, which may then be targeted for degradation in lysosomes or recycled back to the membrane.

Signaling Pathway Desensitization with Clathrin-Coated Vesicles

  • Processes of signaling pathway desensitization involve clathrin-coated vesicles.

  • Arrestins are cytoplasmic proteins that interact with the phosphorylated intracellular loops of G protein-coupled receptors (GPCRs).
      - Process:
        - Upon GPCR activation, phosphorylation occurs, which arrestins recognize.
        - Arrestins then bind to the receptors at the plasma membrane, blocking interactions with G proteins, effectively shutting off the signaling pathway.
        - Arrestins undergo conformational changes that expose clathrin-binding sites, allowing for tethering to clathrin-coated pits and subsequent endocytosis.

Summary of the Secretory Pathway

  • Proteins with a signal sequence (ss) are directed towards the ER where vesicles bud off and move to the cis Golgi.

  • Cisternal maturation within the Golgi apparatus leads to sorting and trafficking of proteins to diverse destinations (primarily plasma membrane or lysosome) without ever entering the cytosol.

Retrograde Transport Returning SNAREs to the ER

  • Retrograde transport from the cis-Golgi back to the ER is crucial for returning SNAREs and any misrouted proteins.

  • This process utilizes distinct mechanisms, employing different coat proteins (COPI) and G-proteins.

Reviewing Protein Targeting Challenges

  • Key challenges in protein targeting include:
      1. Accurately directing proteins to specific areas within the cell.
      2. Translocating hydrophilic proteins across hydrophobic membranes.