Bio Exam 4 Summary PDF

**LEC 14** **Proteins are Synthesized by Ribosomes** - **Mammalian Cells:** Contain up to 10,000 proteins, with the majority synthesized by free cytosolic ribosomes. Around 1/3 are synthesized by ribosomes on the ER membrane. **Synthesis of Proteins on Membrane-Bound vs Free Ribosomes**...

**LEC 14** **Proteins are Synthesized by Ribosomes** - **Mammalian Cells:** Contain up to 10,000 proteins, with the majority synthesized by free cytosolic ribosomes. Around 1/3 are synthesized by ribosomes on the ER membrane. **Synthesis of Proteins on Membrane-Bound vs Free Ribosomes** - **On Rough ER (Membrane-Bound Ribosomes):** - Secreted, Integral & soluble proteins - **On Free Ribosomes:** - Cytosolic peripheral membrane proteins - Nuclear proteins - Proteins targeted to mitochondria, chloroplasts, and peroxisomes **Protein Sorting Mechanisms** - **Pulse-Chase Experiment:** - Radiolabeled amino acids like 35S-methionine are used to track protein synthesis over time. - - Radioactive labeling during a short pulse (3 minutes) and the chase period (varied) allows researchers to track protein movement and destination. - - **GFP-Based Protein Tracking:** - Temperature-sensitive mutants, showing how proteins accumulate or move in response to temperature changes. - **Subcellular Fractionation and Differential Centrifugation:** - Isolate different cellular components to study protein sorting and trafficking. - **Genetic Mutants:** - Studies on yeast (Saccharomyces cerevisiae) help identify mutations affecting protein sorting, with the Nobel Prize in 2013 recognizing these discoveries. **Signal-Based Targeting** - **Signal Sequence Targeting:** - Proteins are tagged with specific signal sequences to direct them to the correct organelles (e.g., ER, mitochondria, peroxisomes, nucleus). - Signal sequences are recognized by receptors, which guide the protein to its destination. - **Example Signal Sequences:** - **ER:** Hydrophobic amino acids at the N-terminus. - **Mitochondria:** Positive charged residues like Arg and Lys. - **Nucleus:** Basic amino acids (e.g., Lys, Arg). - **Peroxisome:** Ser-Lys-Leu at the C-terminus. **Endomembrane System & Secretory Pathway** - **Endomembrane System:** Comprises the ER, Golgi complex, endosomes, lysosomes, vacuoles, and secretory vesicles. - **Biosynthetic Pathway:** Involves synthesis, modification, and transport of proteins. - **Secretory Pathway:** Involves secretion of proteins from the cell, either constitutively (constant) or regulated (stimulated). - **Vesicle-Based Trafficking:** - Vesicles transport proteins between compartments, often utilizing motor proteins and cytoskeletal tracks. **The Endoplasmic Reticulum** - **Smooth ER:** - Involved in steroid hormone synthesis, detoxification, and calcium sequestration. - Found in cells like Leydig cells (steroid hormone synthesis) and liver cells (detoxification). - **Rough ER:** - Connected to the outer nuclear membrane, where ribosomes synthesize secretory and membrane-bound proteins. - Plays a key role in membrane biosynthesis and protein modification. **Membrane Biosynthesis in the ER** - Membrane lipids and proteins are synthesized in the ER. - **Integral membrane proteins:** Active site faces the cytosol. - Membranes are asymmetric with distinct cytosolic and luminal/extracellular faces, established in the ER. **Evidence for Secretory Proteins in the ER Lumen** - Radiolabeled proteins are synthesized and tracked. - Subcellular fractionation isolates microsomes (vesicles derived from ER). - **Pulse-Chase Experiment**: Proteins are protected inside microsomes from protease digestion unless detergent is used, indicating the proteins reside in the ER lumen post-synthesis. **Evidence for Secretory Proteins in the ER Lumen After Synthesis:** 1. **Cotranslational Translocation:** - **Evidence:** Proteins are incorporated into the ER membrane (microsomes) while still bound to the ribosome, during translation. - **Mechanism:** As the nascent polypeptide is being synthesized, its signal sequence is recognized by the **Signal Recognition Particle (SRP)**, which temporarily halts translation and directs the ribosome to the ER membrane. 2. **Pulse-Chase Experiment:** - Radioactively labeled proteins are tracked as they move through the ER. The radioactivity (indicating protein synthesis) can be found inside the ER lumen, confirming that secretory proteins are being inserted cotranslationally. **Synthesis of Secretory Proteins on the Rough ER:** 1. **Step-by-Step Process of Cotranslational Translocation:** 1. **Translation begins** with the formation of a signal sequence (6-12 hydrophobic amino acids at the N-terminus) of the nascent protein. 2. The **Signal Recognition Particle (SRP)** binds to the signal sequence and temporarily halts translation. 3. The **SRP** then guides the ribosome to the **SRP receptor** on the ER membrane, where translation resumes through a **translocon pore**. 4. The signal sequence is inserted into the translocon, and translation resumes, pulling the polypeptide chain through the translocon into the ER lumen. 5. The **signal peptidase** cleaves the signal sequence, leaving the mature protein in the ER lumen. 6. **Chaperones** like **BiP** bind to the nascent protein in the lumen, ensuring proper folding. 2. **Role of BiP:** 1. BiP (a member of the **Hsp70 chaperone** family) assists in protein folding and prevents the translocon pore from opening prematurely when bound to ADP. It has **dual roles** in protein folding, binding tightly to the nascent protein when in its ADP-bound state. **Synthesis of Integral Membrane Proteins:** 1. **Membrane Insertion:** - For **integral membrane proteins**, specific signal sequences direct these proteins to the ER membrane where they are inserted during translation. The orientation of the protein is carefully maintained: the end facing the ER lumen becomes the extracellular space (or the lumen of vesicles like the Golgi). 2. **Single-Pass Transmembrane Proteins:** - **Mechanism:** - A **signal sequence** starts the process, followed by a **stop-transfer sequence** (hydrophobic residues) which halts translocation and anchors the protein in the ER membrane. - Translation continues until the C-terminus of the protein is synthesized, with the N-terminus facing the ER lumen. 3. **Multipass Transmembrane Proteins:** - **Insertion Process:** Multiple hydrophobic regions of the protein pass through the translocon sequentially, with each new transmembrane segment oriented oppositely to the previous one. **Processing of Newly Synthesized Proteins in the ER:** 1. **Proteolytic Cleavages:** - Proteins undergo **cleavage** by enzymes such as **signal peptidase** to remove signal sequences or unnecessary regions. 2. **Disulfide Bond Formation:** - **Disulfide bonds** between cysteine residues are formed in the ER, which stabilize protein structures. This process is facilitated by **protein disulfide isomerase** (PDI). 3. **Glycosylation:** - Many proteins are modified by the addition of carbohydrate groups (**glycosylation**) in the ER. This process aids in proper protein folding and stability, and glycoproteins are involved in cell-cell interactions, adhesion, and signaling. 4. **Chaperone-assisted Folding:** - **BiP** and other chaperones help proteins fold correctly in the ER, ensuring that they maintain their native structures. **Quality Control in the ER:** 1. **Glycoprotein Modification:** - **Calnexin**, an ER chaperone, binds glycoproteins that retain a single glucose moiety, ensuring they are correctly folded. If a protein is misfolded, it cycles back for further modifications until properly folded or it is targeted for degradation. 2. **ER-associated Degradation (ERAD):** - Misfolded proteins are poly-ubiquitinated and sent to the **proteasome** for degradation. A key example is the CFTR ion channel, where mutant versions are degraded by ERAD. **Unfolded Protein Response (UPR):** 1. **Mechanism:** - When unfolded proteins accumulate in the ER, the **UPR** is activated. Sensors like **PERK** and **ATF6** initiate a response that: 1. Reduces protein synthesis. 2. Increases the production of **chaperones** to assist in protein folding. 3. Initiates degradation pathways for misfolded proteins to prevent cellular damage. 2. **Function of BiP in UPR:** - **BiP** maintains the inactivity of UPR sensors until the accumulation of misfolded proteins triggers its release and activation of the UPR signaling pathways. - **LEC 15** **Synthesis of Proteins on Membrane-Bound vs Free Ribosomes** - **1/3 of human proteome synthesized on rough ER** - Secreted proteins - Integral membrane proteins - Soluble proteins of organelles - **Free ribosomes** - Cytosolic proteins - Cytosolic peripheral membrane proteins - Nuclear proteins - Proteins targeted to mitochondria, chloroplasts, and peroxisomes **Topological Equivalency** - Lumen of ER = Lumen of Golgi = Extracellular Space - Lumen of ER = inside vesicle - Lumen of Golgi = extracellular space **Proteins Modifications in ER and Golgi** - **1. Glycosylation** - covalent addition of carbohydrates - **2. Formation of Disulfide Bonds** in ER - **3. Proper Folding of Polypeptide Chains** and assembly of multi-subunit proteins - **4. Specific Proteolytic Cleavages** in ER, Golgi, and secretory vesicles **Quality Control in ER: Protein Folding** - Glycoproteins modified, retaining a single glucose moiety - Glucose recognized and bound by ER chaperone **calnexin** - **Glucosidase II** removes remaining glucose - **UGGT** determines if properly folded - Hydrophobic residues for detection - If protein is misfolded: - Glucose added back for another cycle - Degraded via proteasome (cytosol) - Exit to biosynthetic pathway **Unfolded Protein Response (UPR)** - **BiP** keeps protein sensors inactive - **Kinase** inactivates translation factors - **Transcription factors** activate transcription - Signaling molecules help manage misfolded proteins **Sorting Signals for Targeting Proteins** - **Signal sequence**: Encoded in the amino acid sequence or attached signals (like carbohydrates) - **Signal receptors**: Can be integral membrane proteins or coat components on budding vesicles **Protein Targeting to Mitochondria and Peroxisomes** - **Unique Features**: Import occurs post-translationally - **Mitochondria**: Import through outer membrane, proteins are unfolded during import - **Peroxisomes**: Proteins are folded **Mitochondria** - Inner and outer membranes with distinct functions - **Outer membrane**: Large pores for molecules to move between cytosol and intermembrane space - **Inner membrane**: Site of aerobic respiration, ATP production - Has its own genome, but most proteins are synthesized in the cytoplasm **Targeting Proteins to Mitochondria** - **Mitochondrial Matrix**: - Enzymes like **Citrate Synthase** and **Isocitrate Dehydrogenase** - **Inner Membrane**: - Proteins like **ATP Synthase Subunits** - **Outer Membrane**: - Proteins like **Mitochondrial Porins** - **Intermembrane Space**: - **Cytochrome C** **Signal Sequences for Targeting to Organelles** - **ER**: 6-12 hydrophobic amino acids at N-terminus - **Mitochondria**: N-terminus with hydrophobic, basic amino acids (Arg, Lys) - **Chloroplast**: N-terminus rich in Ser, Thr - **Peroxisome**: C-terminus with Ser-Lys-Leu - **Nucleus**: Internal sequence with basic amino acids **Targeting to Mitochondria** - **Matrix Targeting Signal**: - At the N-terminus (20-50 amino acids long) - Contains: - Hydrophobic amino acids - Positively charged basic amino acids (Arg, Lys) - Hydroxylated amino acids (Ser, Thr) - Lacks negatively charged residues (Asp, Glu) - **Amphipathic alpha-helix**: Hydrophobic amino acids on one side and hydrophilic (basic) on the other **Is protein translocation to the mitochondria "co-translational" or "post-translational"?** - **Post-translational**: Proteins are synthesized in the cytosol and imported into the mitochondria in an unfolded state with the help of chaperones. **Protein Targeting to the Mitochondrial Matrix** 1. **Synthesis in the cytosol**: Proteins are synthesized by ribosomes in the cytosol. - They must be unfolded and are kept in this state by chaperones (Hsp70, Hsp90) using ATP. 2. **Binding to Outer Membrane Receptor**: The protein binds to Tom20/22 receptors on the outer mitochondrial membrane via a matrix-targeting sequence (MTS). 3. **Insertion into Outer Membrane Translocon (Tom40)**: The MTS inserts into the Tom40 translocon, allowing the protein to pass through. 4. **Translocation to Inner Membrane**: The protein moves through Tom40 into the inner membrane translocon (Tim23/17). 5. **Matrix Import**: Matrix Hsp70 helps move the protein into the matrix, and the MTS is cleaved by a protease. 6. **Folding**: Hsp70 releases the protein in the matrix, allowing it to fold into its active conformation, sometimes with the help of chaperonins. **Energy Inputs for Mitochondrial Protein Import** - **Cytosol**: ATP hydrolysis by Hsp70 to keep the protein unfolded. - **Matrix**: ATP hydrolysis by matrix Hsp70 to pull the protein into the matrix. - **Proton-Motive Force (H+ Gradient)**: The electrochemical gradient across the inner membrane drives protein import into the matrix. **Import of Inner Membrane Proteins** - **Stop-Transfer Anchor Sequence**: Proteins that require both an N-terminal MTS and an internal hydrophobic sequence. - **Process**: The hydrophobic sequence blocks further transfer through Tim23/17, and the protein is inserted laterally into the membrane. **Import of Multi-Pass Transmembrane Proteins** - **Multiple Targeting Sequences**: These proteins have no N-terminal MTS but multiple internal mitochondrial targeting sequences. - **Process**: They pass through Tom40, then through Tim22/54 for membrane insertion. **Targeting to the Intermembrane Space** 1. **Matrix Targeting Sequence**: Leads the protein to the matrix and is cleaved. 2. **Second Hydrophobic Sequence**: Blocks full translocation into the matrix, causing the protein to insert into the inner membrane. **Peroxisomes** - **Function**: Peroxisomes are involved in oxidative metabolism, such as breaking down long-chain fatty acids and hydrogen peroxide (via catalase). - **Diseases**: Zellweger syndrome (lack of peroxisomal enzymes) and Adrenoleukodystrophy (lack of fatty acid transporter). **Protein Targeting to Peroxisomes** 1. **Peroxisomal Targeting Sequence (PTS1)**: Ser-Lys-Leu (SKL) at the C-terminus directs proteins to the peroxisome. 2. **Receptor**: Pex5 binds to the PTS1 sequence and facilitates translocation. 3. **Translocation**: Proteins can be imported in their folded state. 4. **Transient Pore Model**: A transient pore is formed for protein import, which is later closed, ensuring controlled entry. **General Mechanism for Protein Targeting** 1. **Signal Sequence**: Directs the protein to the correct destination. 2. **Receptor**: Specific to the target organelle, binds to the signal sequence. 3. **Translocation Channel**: Allows the protein to pass through the membrane. 4. **Energetic Coupling**: Translocation is driven by GTP or ATP hydrolysis to ensure unidirectional movement. **LEC 16** **Nucleus Overview** - **Primary Function**: The nucleus is often referred to as the \"headquarters\" of the cell, as it contains the cell\'s genetic material (DNA) and regulates gene expression and cellular functions. **Nucleolus** - **Function**: This is the site for the synthesis of ribosomal RNA (rRNA) and assembly of ribosomal subunits. **Nuclear Matrix** - **Composition**: Made of intermediate filaments known as lamins and other associated proteins. - **Function**: Provides structural support and helps maintain the shape and integrity of the nucleus. **Nuclear Envelope** - **Structure**: Double membrane system consisting of: - **Inner membrane** - **Outer membrane** (continuous with the endoplasmic reticulum) - **Nuclear Pore Complexes (NPCs)**: Embedded in the nuclear envelope, they regulate the transport of molecules into and out of the nucleus. **Chromatin** - **Definition**: Complex of DNA and associated proteins (e.g., histones) that forms the structural basis of chromosomes within the nucleus. - **Types**: - **Euchromatin**: Loosely packed and transcriptionally active. - **Heterochromatin**: Densely packed and transcriptionally inactive (silent genes). **Nucleoplasm** - **Description**: The semi-fluid substance within the nucleus, excluding the nucleolus and chromatin. **Nuclear Lamina** - **Function**: Supports the nuclear envelope and maintains nuclear structure. - **Composition**: Primarily consists of lamins, which are a type of intermediate filament protein. - **Regulation**: Integrity is maintained through phosphorylation and dephosphorylation cycles. - **Clinical Relevance**: - **Hutchinson-Gilford Progeria Syndrome (HGPS)**: Caused by mutations in lamin A/C, leading to premature aging. - **Leukodystrophy**: Caused by mutations in lamin B, affecting myelin formation. **Nuclear Pore Complex (NPC)** - **Structure**: Composed of \~30 proteins called nucleoporins, forming an octagonal ring-like structure with an aqueous central pore. - **Dimensions**: Large structure (\~60,000-80,000 kDa) spanning both membranes of the nuclear envelope. - **Functions**: - **Transport**: Allows diffusion of small molecules (\