Protein Synthesis and Sorting Mechanisms

Proteins Synthesized by Ribosomes

• Mammalian cells contain up to 10,000 proteins
• Majority are synthesized by free cytosolic ribosomes
• About 1/3 are synthesized by ribosomes on the endoplasmic reticulum (ER) membrane

Synthesis of Proteins on Membrane-Bound vs Free Ribosomes

• Rough ER (Membrane-Bound):
  • Secretory, integral, and soluble proteins
• Free Ribosomes:
  • Cytosolic peripheral membrane proteins
  • Nuclear proteins
  • Proteins targeted to mitochondria, chloroplasts, and peroxisomes

Protein Sorting Mechanisms

• Pulse-Chase Experiment:
  • Radiolabeled amino acids (e.g., 35S-methionine) are used to track protein synthesis over time
  • Tracking protein movement and destination
• GFP-Based Protein Tracking:
  • Temperature-sensitive mutants show protein accumulation or movement in response to temperature changes
• Subcellular Fractionation & Differential Centrifugation:
  • isolating different cellular components to study protein sorting and trafficking
• Genetic Mutants:
  • Studies on yeast help identify mutations that affect protein sorting

Signal-Based Targeting

• Signal Sequence Targeting:
  • Proteins are tagged with specific signal sequences for correct organelle delivery (e.g., ER, mitochondria, peroxisomes, nucleus)
  • Signal sequences are recognized by receptors guiding proteins to their destinations
  • Example sequences:
    • ER: Hydrophobic amino acids at the N-terminus
    • Mitochondria: Positively charged amino acids (e.g., Arg, Lys)
    • Nucleus: Basic amino acids (e.g., Lys, Arg)
    • Peroxisome: Ser-Lys-Leu at the C-terminus

Endomembrane System & Secretory Pathway

• Comprises ER, Golgi complex, endosomes, lysosomes, vacuoles, and secretory vesicles
• Biosynthetic Pathway: Involves protein synthesis, modification, and transport
• Secretory Pathway: Involves secretion of proteins from the cell, either constitutively or regulated
• Vesicle-Based Trafficking: Vesicles transport proteins between compartments, utilizing motor proteins and cytoskeletal tracks

Membrane Biosynthesis in the ER

• Membrane lipids and proteins are synthesized in the ER
• Integral membrane proteins have active sites facing the cytosol
• Membranes are asymmetric with distinct cytosolic and luminal/extracellular faces, established in the ER
• Evidence for Secretory Proteins in the ER Lumen:
  • Radioactive proteins and subcellular fractionation (tracking via microsomes) prove proteins reside in the ER lumen post-synthesis

Synthesis of Secretory Proteins on the Rough ER

• Step-by-Step Process of Cotranslational Translocation: -Involves translation, signal sequence recognition, SRP and receptor interaction, insertion into translocon, protein translocation into the lumen, signal sequence cleavage, and protein folding

Processing of Newly Synthesized Proteins in the ER

• Proteolytic Cleavages: Removal of signal sequences or unwanted regions
• Disulfide Bond Formation: Provides protein structure stability
• Glycosylation: Addition of carbohydrates to proteins aiding proper folding and stability (involved in cell-cell interactions, adhesion, and signaling)
• Chaperone-Assisted Folding: BiP and other chaperones help proteins fold correctly

Quality Control in the ER

• Glycoprotein Modification: Calnexin ensures proper glucose modification and folding, targeted degradation for misfolded proteins
• ER-Associated Degradation (ERAD): Misfolded proteins are degraded in the proteasome (cytosol)

Unfolded Protein Response (UPR)

• Activated when unfolded proteins accumulate in the ER
• Sensors like PERK and ATF6 initiate a response that reduces protein synthesis, increases chaperone production, and initiates degradation
• BiP maintains UPR sensor inactivity until misfolded protein triggers release and activation
• BiP helps fold proteins, prevent translocon pore opening prematurely.

Synthesis of Proteins on Membrane-Bound vs Free Ribosomes

-About 1/3 of human proteins synthesized on rough ER -Secretory 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

Protein Modifications in ER & Golgi

• Glycosylation
• Disulfide Bond Formation
• Proper Protein Folding
• Specific Proteolytic Cleavages

Quality Control in ER: Protein Folding

• Glycoproteins modified, retaining single glucose moieties
• Glucose recognition & binding by ER chaperones (calnexin)
• Glucosidase II removing remaining glucose
• UGGT assesses correct folding
• Hydrophobic residues for detection
• If misfolded: -Glucose added back -Degraded via proteasome -Exit to biosynthetic pathway

Protein Modification Issues: The UPR

• BiP maintains sensor inactivity until misfolded protein accumulation
• Kinases inactivate translation factors
• Transcription factors activate transcription
• Signal molecules help manage misfolded proteins

Protein Targeting to Mitochondria & Peroxisomes

• Import occurs post-translationally
• Mitochondria: Import through the outer membrane
• Peroxisomes: Proteins already folded
• Targeting involved specific signals (e.g., 6-12 hydrophobic amino acids).
• Disorders result in problems with protein function in locations

Targeting to Mitochondria

• Matrix Targeting signals, contains hydrophobic amino acids and positively charged ones (Arg, Lys)
• Import through the outer membrane as unfolded, then through the inner membrane via a translocon pore
• Help from chaperones (Hsp70,Hsp90) needed
• Targeting sequences important

Targeting to Peroxisomes

• Peroxisomal targeting sequence (PTS1), usually at the C-terminus (often Ser-Lys-Leu)
• Receptor Pex5 binds to PTS1, facilitating translocation
• Proteins are often imported in their folded state
• Transport to Intermembrane Space
• Matrix → Inner Membrane
• Second Hydrophobic Sequence (blocks translocation)
• Causes insertion into membrane

General Mechanism for Protein Targeting

• Signal Sequence: Directs protein to correct location
• Receptor: Binds to specific signal sequence for organelle
• Translocation Channel: Allows protein passage through membrane
• Energetic Coupling: GTP/ATP drives unidirectional movement

Nucleus Overview

• Primary Function: Contains DNA, regulates gene expression, and cellular functions
• Nucleolus: rRNA synthesis and ribosomal subunit assembly
• Nuclear Matrix: Structural support for nucleus, made of lamins
• Nuclear Envelope: Double membrane system

Nuclear Pore Complexes (NPCs)

• Dimensions: Large structure spanning both membranes of nuclear envelope
• Functions: Transport of small and large molecules

Trafficking Through NPCs

• Exported molecules: mRNA, tRNA, Proteins
• Imported molecules: Proteins, transcription factors, enzymes

mRNA Processing Overview

• 5' Methylguanosine Cap Addition: Protects from degradation and helps ribosome binding
• Poly A Tail Addition: Increases stability and aids export from nucleus
• Splicing: Removal of introns, joining of exons, forms mature mRNA
• mRNA is exported through the nuclear pore complex

5' Methylguanosine Cap

• Prevents degradation, aids in mRNA transport, and is involved in translation initiation

Structure of Mature Eukaryotic mRNA

• Coding and noncoding regions (5'UTR and 3'UTR).
• 5' cap, Poly A tail.

Fate of mRNA

• After processing, mRNA can be stored, translated, or decayed. Nuclear envelope breaks down during mitosis, and mRNA exports through nuclear pore complex (NPC).

How mRNAs are Exported from the Nucleus

• mRNA is bound by proteins (mRNP)
• Forms co-transcriptionally

Protein Import / Export Mechanisms

• Nuclear Localization Signal (NLS): Short amino acid sequence that targets proteins; Importin receptor facilitates transport
• Nuclear Export Signal (NES)
• Importin, Exportin
• Ran protein, GTP, GDP, and GTP hydrolysis
• Nuclear Pore Complexes (NPC) aid transport through the nuclear membrane
• Energy input via GTP hydrolysis regulates transport.

Trafficking & Endomembrane System

• Interconnected membrane systems, including ER, Golgi, lysosomes, vesicles, endosomes.
• Secretory pathway: Movement from donor compartment to acceptor compartment via vesicle budding followed by fusion

Breakdown of Secretory Pathway

• Protein synthesis in the ER: folding, modification (glycosylation, disulfide bonds)
• Anterograde transport to Golgi: Vesicles fuse with cis Golgi
• Retrograde transport; recover ER proteins back to cis Golgi
• Cisternal maturation: Progression from cis to medial to trans Golgi
• Further modifications within the Golgi
• Vesicles to final destinations

Experimental Methods

• GFP-Based Tracking (temp sensitive mutants)
• Compartment-specific modifications (e.g. yeast genetic mutants, for vesicular transport)

Mechanisms of Vesicle Formation

• GTPase Activation (GDP to GTP)
• Priming complex formation (coat-GAP interaction)
• Cargo Incorporation (interaction with coat proteins)

G Proteins and Coats

• Sar1 (COPII) for ER to Golgi transport, ARF for COPI and Clathrin mediated transport

Vesicle Targeting and Fusion

• Rab GTPases for docking
• SNARE Proteins (v-SNAREs and t-SNAREs): mediate fusion

Transport Vesicle Fusion with Target Membrane

• Docking (Rab proteins & lipid anchors)
• v-SNARE and t-SNARE interaction: forming complexes
• Membrane fusion through hydrophobic contact
• Cargo release

Dissociation of SNARE Complexes

• NSF and a-SNAP bind to the SNARE complex
• ATP hydrolysis resets the components

Protein-Coated Vesicles and Transport Types

• COPII-coated vesicles: ER to ERGIC/Golgi
• COPI-coated vesicles: Retrograde transport
• Clathrin-coated vesicles: TGN to endosomes/lysosomes

Golgi Complex - Function and Transport

• Protein modification (e.g., glycosylation)
• Sorting and distribution

Anterograde Transport and Retrograde Transport

• Movement of proteins from cis to trans Golgi compartments
• Return of Golgi enzymes or ER proteins to the ER

This quiz explores the intricate processes of protein synthesis in mammalian cells, focusing on the roles of free cytosolic ribosomes and membrane-bound ribosomes on the rough endoplasmic reticulum. It also covers various protein sorting mechanisms such as the pulse-chase experiment and GFP-based tracking. Test your knowledge on how proteins are synthesized, sorted, and directed to their functional locations within the cell.