Protein Targeting and Secretory Pathways
Protein Targeting and Secretory Pathways
Learning Objectives (ILOs)
Understanding the differences between prokaryotic and eukaryotic cells, including their structural and functional characteristics.
Understanding cell compartmentalization and the importance of organelles in eukaryotic cells.
Gaining insights into the endomembrane system, its components, and their interconnected functions.
Understanding transport signals and pathways that direct proteins to their correct destinations.
Identifying the molecular machinery involved in transport, such as coat proteins, SNAREs, and Rabs.
The Central Dogma of Life
[Illustration of the central dogma - needs further detail from the slides]: The central dogma describes the flow of genetic information within a biological system. DNA is transcribed into RNA, which is then translated into protein. This process is fundamental to all living organisms.
Prokaryotic vs. Eukaryotic Cells
Eukaryotic Cells:- Plant and animal cells.
Features: Nucleus (containing DNA organized into chromosomes), nucleolus, chromosomes, cytosol, cytoplasm, early endosome, Golgi apparatus, late endosome, lysosome, endoplasmic reticulum (ER - both rough and smooth), secretory vesicle, cisternae, plasma membrane, ribosomes (both free and ER-bound), mitochondrion, vacuole, chloroplast (in plant cells), plasmodesma (in plant cells).
Eukaryotic cells are generally larger and more complex than prokaryotic cells. They exhibit a high degree of internal organization through compartmentalization.
Prokaryotic Cells:- Bacterial cells (bacillus type).
Features: Cell wall, plasma membrane, ribosomes, cytoplasm, chromosome (circular DNA), pili, capsule, mesosome, flagella.
Prokaryotic cells lack a nucleus and other membrane-bound organelles. Their genetic material is located in the cytoplasm.
Compartmentalization:- Organelles within eukaryotic cells create specialized compartments, allowing for efficient and regulated biochemical processes.
Compartmentalization increases efficiency by concentrating enzymes and substrates within specific organelles.
Each organelle provides a unique environment that supports specific biochemical reactions.
Endomembrane System
The cell is subdivided into different organelles, which are interconnected through the endomembrane system.
Specialization of function occurs within these organelles; for example, the ER is involved in protein synthesis and folding, while the Golgi apparatus modifies and sorts proteins.
Partnership between organelles facilitates function, allowing for coordinated cellular activities.
The endomembrane system includes the endoplasmic reticulum (ER), Golgi apparatus, lysosomes, endosomes, and the plasma membrane.
Camillo Golgi
Camillo Golgi (1843-1926) discovered the Golgi apparatus through his work on the nervous system.
The Golgi apparatus is a basket-like network surrounding the nucleus in Purkinje cells, observed through metallic impregnation, a staining technique he developed.
Golgi's discovery of the Golgi apparatus was initially controversial but later confirmed and recognized with the Nobel Prize in Physiology or Medicine in 1906.
Cargo Transportation
Proteins are transported via different mechanisms to reach their correct locations within the cell:
Across membranes: Proteins can be directly translocated across organelle membranes.
Through nuclear pores: Proteins enter and exit the nucleus through nuclear pore complexes.
By vesicles: Proteins are packaged into vesicles that bud off from one organelle and fuse with another.
Key organelles involved: Chloroplast, mitochondrion, ER, nucleus, Golgi, endosomes, and lysosomes.
Different Routes
Proteins take various routes within the cell, depending on their function and destination:
Plasma membrane: Proteins destined for the cell surface.
Secretory vesicle: Proteins that are secreted from the cell.
Cytosol: Proteins that function within the cytoplasm.
Early endosome: Proteins involved in endocytosis.
Late endosome: Proteins destined for degradation.
Golgi apparatus: Proteins that are modified and sorted.
Lysosome: Proteins that are involved in degradation.
Endoplasmic reticulum (ER): Proteins that are synthesized and folded.
Cisternae: Proteins within the Golgi apparatus.
Protein Signals
Proteins contain conserved amino acid sequences that act as molecular addresses, directing them to specific locations within the cell.
Signal Sequences Examples:
Import into ER: H3N-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-Phe-Gln-
Retention in the lumen of the ER: *
-Lys-Asp-Glu-Leu-COO- commonly referred to as KDEL* (Lysine-Aspartic acid-Glutamic acid-Leucine)Import into mitochondria: H3N-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu-Leu-
Import into the nucleus: *
-Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-*Import into peroxisomes: *
-Ser-Lys-Leu-* (Serine-Lysine-Leucine)
Positively charged amino acids are shown in red, and negatively charged amino acids in blue.
An extended block of hydrophobic amino acids is shown in green.
*H3N indicates the N-terminus of a protein; COO- indicates the C-terminus.
Signal sequences are typically 15-60 amino acids long and are often located at the N-terminus of the protein.
Signal Peptide Structure
N-region: positive region often containing Arginine (Arg) and Lysine (Lys), which helps in the interaction with the negatively charged phospholipids in the ER membrane.
h-region: Hydrophobic region, 7-15 residues long, rich in Leucine (Leu), which facilitates insertion into the lipid bilayer.
c-region: uncharged region, 3-7 residues long, containing Alanine (Ala)-X-Ala-Glu-Ala, where X is any amino acid residue. This region is important for signal peptidase recognition.
Cleavage site between P1 and P'1 positions, where signal peptidase cleaves the signal peptide from the mature protein.
Examples: Signal Peptide and Signal Peptidase (SPase)
Signal peptidase (SPase) cleaves signal sequences in the ER lumen, releasing the mature protein.
Examples of signal sequences and their corresponding proteins:
MKWVTFISLLFSSAYS (16aa) - Albumin: Serum albumin is a major protein in blood plasma.
MKCLLYLAFLFIGVNC - VSV-G (16aa): Vesicular Stomatitis Virus G protein, a viral glycoprotein.
MGQIVTMFEALPHIIDEVINIVIIVLIIITSIKAVYNFATCGILALVSFLFLAGRSCG-LCMV GP-C (58aa): Lymphocytic choriomeningitis virus glycoprotein.
MPNHQSGSPTGSSDLLLSGKKQRPHLALRRKRRREMRKIN - MMTV Rem (98aa): Mouse mammary tumor virus Rem protein.
MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS - Prl (30aa): Rat preprolactin, a precursor to the hormone prolactin.
Transport Molecular Machinery
Secretory pathways unite at least 10 cellular compartments, including the ER, Golgi, endosomes, and lysosomes.
Direction and specificity are determined by receptor : ligand interactions, ensuring that proteins are transported to the correct destination.
Vesicles Mediate Cargo Transport
Vesicles transport cargo between organelles, budding from one organelle and fusing with another.
Specific donor:acceptor vesicle interactions ensure correct targeting, mediated by coat proteins, SNAREs, and Rabs.
Vesicles bud from:
ER cisternae: Transporting newly synthesized proteins to the Golgi.
Golgi apparatus: Transporting modified proteins to other organelles or the plasma membrane.
Vesicle Coat Proteins
Vesicles have coat proteins that facilitate their formation and targeting. These proteins help to deform the membrane, select cargo, and target the vesicle to the correct destination.
Types of Coat Proteins:
Clathrin: Involved in transport from the Trans-Golgi Network (TGN) to endosomes and in endocytic transport. Clathrin-coated vesicles are involved in receptor-mediated endocytosis and transporting lysosomal enzymes from the TGN to endosomes.
COPI: Involved in retrograde transport from the Golgi to the ER and within Golgi compartments. COPI-coated vesicles retrieve ER-resident proteins that have escaped to the Golgi and transport proteins between Golgi cisternae.
COPII: Involved in transport from the ER to the Golgi. COPII-coated vesicles are responsible for the selective export of cargo molecules from the ER.
Different Coat Proteins
Clathrin:
Trans-Golgi Network (TGN) to endosome and endocytic transport
COPI:
Golgi to ER and within Golgi compartments
COPII:
ER to Golgi Vesicles – specific transport
Summary 1
Bidirectional transport between ER and Golgi is essential for maintaining the proper composition of these organelles.
Forward pathway: Secretory proteins move from ER to Golgi for further processing and sorting.
Retrieval pathway: ER-resident proteins with KDEL signal sequences are retrieved from the Golgi back to the ER by KDEL receptor. This ensures that ER-resident proteins are not lost from the ER.
Vesicular tubular clusters facilitate this transport, acting as transport intermediates between the ER and Golgi.
Vesicle Shapes
Different coat proteins result in different vesicle shapes, influencing their function and targeting.
Clathrin-coated vesicles: Spherical shape.
COPI-coated vesicles: Bud-shaped.
COPII-coated vesicles: Tubular shape.
Size is approximately , although the size can vary depending on the cargo and coat protein.
Clathrin
Involved in transport from the Trans-Golgi Network (TGN) to endosomes and endocytic transport.
Consists of heavy and light chains that assemble to form a polyhedral lattice around the vesicle.
Size is approximately . Clathrin-coated vesicles are involved in various cellular processes, including receptor-mediated endocytosis and protein trafficking.
Coat Assembly and Cargo Selection
Coat assembly and cargo selection are critical steps in vesicle formation, ensuring that the correct cargo is packaged into the vesicle and transported to the correct destination.
Cargo adaptor proteins bind to cargo receptors, recognizing specific signal sequences on the cargo molecules.
Clathrin binds to the adaptor protein, forming a coat around the vesicle.
Bud formation, where the membrane begins to curve and form a vesicle.
Vesicle formation, where the vesicle buds off from the donor organelle.
Uncoating occurs, releasing the naked transport vesicle in the cytosol, allowing it to fuse with the target organelle.
COPI: Golgi to ER and within Golgi compartments
Arranged in pentagons and hexagons, forming a coat around the vesicle.
COPII: ER to Golgi
Cage-like structure that helps to deform the ER membrane and select cargo molecules for transport to the Golgi.
Approximately in diameter.
Summary 2
Vesicles are delineated by a membrane and filled with cargo, which can include a variety of molecules.
Cargo includes:
Secretory proteins: Proteins that are secreted from the cell.
Lysosomal enzymes: Enzymes that degrade macromolecules in the lysosome.
Cell surface and ECM components: Proteins and other molecules that are found on the cell surface or in the extracellular matrix.
Vesicle membrane composition dictates its fate, including cargo composition and target compartment. The lipid and protein composition of the vesicle membrane are important for targeting and fusion.
Accessory Proteins - v-SNAREs and t-SNAREs
Mediate specificity in vesicle targeting and fusion, ensuring that vesicles fuse with the correct target compartment.
v-SNAREs are located on the vesicle membrane, while t-SNAREs are located on the target organelle membrane.
These proteins facilitate docking and fusion of the vesicle with the correct target compartment by forming a SNARE complex that brings the vesicle and target membranes into close proximity.
Accessory Proteins - Rabs
GTPases that also provide specificity in vesicle targeting and fusion. Rabs act as molecular switches, cycling between an active GTP-bound state and an inactive GDP-bound state.
Rab-GTP binds to Rab effectors on the target membrane, facilitating docking. Rab effectors are proteins that interact with Rab-GTP and help to tether the vesicle to the target membrane.
Guanine-nucleotide-exchange factor (GEF) activates Rab by converting Rab-GDP to Rab-GTP. GEFs are localized to specific organelles and help to ensure that Rabs are activated at the correct location.
GDP dissociation inhibitor (GDI) regulates Rab activity by binding to Rab-GDP and preventing it from interacting with GEFs.
GTP hydrolysis leads to membrane fusion, allowing the vesicle to release its cargo into the target compartment.
Different Rabs
Different Rab proteins are localized to different organelles, contributing to the specificity of vesicle trafficking.
Rab1: ER and Golgi complex
Rab2: cis Golgi network
Rab3A: synaptic vesicles, secretory granules
Rab4/Rab11: recycling endosomes
Rab5A: plasma membrane, clathrin-coated vesicles, early endosomes
Rab5C: early endosomes
Rab6: medial and trans Golgi cisternae
Rab7: late endosomes
Rab8: early endosomes
Rab9: late endosomes, trans Golgi network
Rabs and Tumor Microenvironment
Rabs are involved in various processes related to tumor development, including:
Migration/invasion: Rabs can regulate the movement of tumor cells.
Proliferation: Rabs can promote the growth of tumor cells.
Apoptosis: Rabs can inhibit programmed cell death in tumor cells.
Endocytosis: Rabs can regulate the uptake of nutrients and growth factors by tumor cells.
Lysosome function: Rabs can regulate the degradation of cellular components in tumor cells.
Tumor initiation: Rabs can contribute to the formation of tumors.
Molecular Specificity
Rab proteins and phosphatidylinositol-3-phosphate (PI(3)P) contribute to molecular specificity during phagosome maturation. This ensures that phagosomes fuse with the correct target organelles to degrade engulfed pathogens and cellular debris.
Rab5 is present on early phagosomes, recruiting proteins involved in phagosome maturation.
Rab7 and PI(3)P are present on late phagosomes, promoting fusion with lysosomes.
Lamp1 is present on phagolysosomes, marking them as mature degradative compartments.
pH decreases as the phagosome matures into a phagolysosome, creating an acidic environment that promotes degradation.
Rabs and SNAREs
Rab-GTP interacts with Rab effectors to facilitate tethering, bringing the vesicle and target membranes into close proximity.
v-SNAREs and t-SNAREs mediate docking and fusion, forming a SNARE complex that promotes membrane fusion.
Vesicles - True Face
Complex protein composition including:
Synaptobrevin: A v-SNARE involved in neurotransmitter release.
SNAP25: A t-SNARE located on the plasma membrane.
Syntaxin: Another t-SNARE located on the plasma membrane.
Rab: A GTPase that regulates vesicle trafficking.
Reminder: Exocytosis and Endocytosis
-(A) exocytosis: the process by which cells release molecules into the extracellular space by fusion of vesicles with the plasma membrane.
-(B) endocytosis: the process by which cells take up molecules from the extracellular space by invagination of the plasma membrane and formation of vesicles.