Unit 5: Protein Distribution and Transport

Unit 5: Protein Distribution and Transport

• Proteins carry out functions determined by genomic DNA information.
• Protein synthesis is the final stage of gene expression.
• mRNA translation is the first step in protein function, demanding folding and processing.
• Gene expression is regulated at the translational level.
• Mechanisms control protein activity and quantity (e.g., differential degradation).
• Eukaryotic cells have membrane-bound organelles in the cytoplasm.
• Protein transport between organelles is a complex process.

Index

• 5.1. Protein synthesis, processing, and regulation
  • mRNA translation
  • Protein folding and processing
  • Regulation of protein function
  • Protein degradation
• 5.2. Endoplasmic reticulum
• 5.3. Golgi apparatus
• 5.4. Vesicle transport mechanism
• 5.5. Lysosomes

mRNA Translation

• Transfer RNA (tRNA)
• Ribosomes
• mRNA organization and translation initiation
• Translation process
• Regulation of translation
• Proteins are synthesized from mRNA
• mRNAs read 5' to 3'
• Polypeptide chains synthesized from amino terminus to carboxyl terminus
• Amino acid (aa) encoded by 3 bases (codon) in mRNA
• tRNA adapters between mRNA and amino acids incorporated into protein
• tRNA structure: 70–80 nucleotides (nt) in length, cloverleaf shape, L-shaped folding, CCA sequence at 3' end for amino acid attachment, anticodon loop for codon binding

Aminoacyl tRNA Synthetases

• Very selective enzymes that attach the appropriate amino acid to its corresponding tRNA.
• Mechanism:
  • Amino Acid + ATP → Aminoacyl-AMP + PPi
  • Aminoacyl-AMP + tRNA → Aminoacyl-tRNA + AMP

Genetic Code

• 64 possible codons
• 61 encode amino acids (aa)
• 3 are stop codons
• Most amino acids encoded by more than one codon (more than 1 tRNA per amino acid)
• Nonstandard codon-anticodon base pairing (wobble) allows for more than one codon to specify the same amino acid.

Ribosomes

• Prokaryotic 70S ribosome: 23S and 5S rRNAs (34 proteins), 16S rRNA (21 proteins)
• Eukaryotic 80S ribosome: 28S, 5.8S, and 5S rRNAs (~46 proteins), 18S rRNA (33 proteins)
• rRNA catalyze peptide bond formation.
• Lack of ribosomal proteins causes decrease, not complete loss of functionality in ribosome function.

Organization of mRNAs and Initiation of Translation

• mRNAs have noncoding untranslated regions (UTRs) at the ends.
• Most eukaryotic mRNAs are mono-cistronic (encode a single protein).
• Prokaryotic mRNAs are often polycistronic (encode multiple proteins).
• AUG codon signifies the start of translation (most bacteria use N-formylmethionine).

Initiation of Translation (Bacteria)

• 30S ribosomal subunit binds IF1 and IF3.
• mRNA, initiator tRNA, and IF2 (bound to GTP) join the complex.
• tRNA binds to the start codon.
• IF1 and IF3 are released.
• 50S subunit associates with the complex, leading to GTP hydrolysis and IF2 release.
• Initiation complex catalyzes peptide bond formation.

Initiation of Translation (Eukaryotes)

• eIF1, eIF1A, and eIF3 bind to the 40S subunit.
• eIF2 (bound to GTP) binds to the initiator methionyl tRNA
• The cap at 5’ end of the mRNA is recognized by eIF4E, forming a complex with eIF4A and eIF4G.
• eIF4A, eIF4B and eIF4G also bind to poly A binding protein
• eIF2 GTP and tRNA combine
• When AUG is recognized, eIF5 causes hydrolysis of the GTP bound to eIF2 and the eIFs are released.
• Facilitating binding of 60S subunit.

Elongation

• Ribosomes have three binding sites: P (peptidyl), A (aminoacyl), and E (exit).

Termination

• Elongation continues until a stop codon (UAA, UAG, or UGA) is reached.
• Release factors recognize the stop codon and terminate protein synthesis.

Regulation of Translation

• Transcription is the primary mechanism of gene expression regulation.
• Translation repressor proteins inhibit translation
• Noncoding microRNAs globally repress translation in response to cellular stress (e.g., starvation, growth factor depletion).

Regulation of eIF4E

• In the absence of growth factors, 4E-BPs bind to eIF4E, preventing its association with eIF4G, thus inhibiting translation.
• Stimulation by growth factors induces phosphorylation of 4E-BPs, causing their dissociation allowing translation to begin.

Protein Folding and Processing

• Chaperones aid protein folding, preventing misfolding and aggregation.
• Diseases stem from protein folding defects.
• Enzymes like protein disulfide isomerase (PDI) catalyze protein folding, notably crucial in secreted and membrane proteins.
• Proteolysis is the removal of initial methionine, additions of chemical groups, and elimination of amino-terminal signal sequences to produce functional proteins, from larger inactive precursor proteins such as insulin, digestion enzymes, and HIV.
• Glycosylation involves adding carbohydrate chains (glycoproteins), with types distinguished by attachment site ( N- or O-linked).
• Proteins can be marked and anchored to the plasma membrane by lipid binding.

Regulation of Protein Function

• Proteins often regulated by small molecules causing conformational changes (allosteric regulation).
• Phosphorylation is a common modification for controlling activity in response to environmental signals.
• Protein-protein interactions regulate protein activity, often involving the formation of protein complexes.

Protein Degradation

• Protein levels are regulated by synthesis and degradation rates.
• Regulatory proteins break down rapidly.
• Defective or damaged proteins are degraded: ubiquitin-proteasome pathway and lysosomal proteolysis.

The Ubiquitin-Proteasome Pathway

• Main route of selective protein degradation.
• Polyubiquitinated proteins are targeted for degradation by the proteasome.

Lysosomal Proteolysis

• Lysosomes contain proteases for degrading proteins, DNA, RNA, and various polymers.
• Lysosomes digest material captured from outside the cell and cellular components.

Endocytosis and Lysosome Formation

• Lysosomes are essential for digesting material from the outside of the cell and from within via endocytosis.
• Endosomes and lysosomes arise from the secretory pathway and the endocytic pathway's intersection.

Early, Recycling, and Late Endosomes

• Early endosomes are formed from endocytosis.
• Material from the outside of a cell is ingested via clathrin-coated vesicles.
• Early endosomes mature into late endosomes, precursors of lysosomes.

Phagocytosis and Autophagy

• Phagocytosis is how a cell engulfs and digests material outside the cell.
• Autophagy is a cell's recycling process where cellular components are engulfed and digested, essential for development and nutrient deprivation.

Smooth Endoplasmic Reticulum (SER) and Lipid Synthesis

• SER is the site of lipid synthesis for cellular membranes.
• Lipids like phospholipids and sterols are synthesized on its cytosolic side, incorporating to the membrane.
• Phospholipids are subsequently translocated to create a uniform bilayer.
• Multiple flippases are required to move phospholipids to opposite positions to create a bilayer.

ER and Protein Secretion - Protein Export and Insertion

• Proteins are translocated into the ER, either co-translationally or post-translationally.
• Proteins can be initially retained in the ER or transported to other organelles like the Golgi, lysosomes or plasma membrane in vesicles.
• Some proteins are directed toward other cellular membranes through the secretory pathway.
• Proteins with internal transmembrane sequences may also be delivered to the ER membrane by alternative pathways.

Golgi Organization and Function

• The Golgi consists of flattened membrane-bound sacs (cisternae), with a cis and trans face.
• Molecules are modified as they travel through the Golgi compartments.
• Golgi involved in glycosylation and lipid metabolism.

Vesicle Transport Mechanism

• Vesicular transport is responsible for carrying material between cellular compartments.
• Vesicles are involved in transport to different cellular locations.
• Coat proteins are involved in vesicle budding and transport.
• SNARE proteins mediate vesicle fusion with target membranes.

Labeling of proteins to target the ER

• Proteins for transport to the ER are identified by a signal sequence. This is incorporated during synthesis and ensures proper positioning.
• The signal sequence allows ribosome binding to ensure proper insertion into the ER membrane.

Protein Folding and Processing in the ER

• The ER assists in protein folding to ensure efficient and functional proteins.
• The ER includes a quality control process to eliminate misfolded proteins (ERAD).
• This process involves chaperones, which help in protein folding, and degradation.

Quality Control in the ER

• Misfolded proteins are recognized and exported from the ER to the cytosol for degradation in the ubiquitin-proteasome pathway.
• Proteins with correct folding remain and mature.

Glycoprotein Folding and Processing in the Golgi Apparatus

• Modifying glycoproteins' carbohydrate regions in the Golgi.
• Enzymes add, remove, and modify sugars.
• Carbohydrate modifications depend on the specific type of protein.

Labeling and Targeting of Lysosomal Proteins by Phosphorylation of Mannose Residues

• Proteins are processed in the Golgi, including those destined for lysosomes.
• The initial reaction often involves removal of mannose residues.
• Unique modifications(e.g., mannose-6-phosphate) are added to specific mannose residues to target to lysosomes.
• Mannose-6-phosphate receptors direct transported proteins to lysosomes.

Proteins Modifications by O-glycosylation

• Some proteins have side chains modified by O-linked oligosaccharides.
• Proteoglycans are an example of proteins extended by long carbohydrate chains, which are essential in the ECM.

Lipid and Polysaccharide Metabolism in the Golgi

• Golgi aids in synthesizing and processing important lipids, such as glycolipids and sphingolipids, and related molecules.
• Glycolipids and sphingolipids are added to proteins and lipids as they proceed through Golgi compartments.
• Processed lipids are properly positioned to be transported in vesicles to other cellular compartments and/or to the cell exterior.

Export of proteins and lipids from the ER

• Proteins and lipids destined for other destinations are packaged in vesicles budding from the ER exit sites (ERESS) to the Golgi intermediary compartment or ERGIC.
• Proteins destined for the Golgi, plasma membranes, and other locations are transported.

Protein Degradation in the cell

• Ubiquitin-proteasome pathway and lysosomal proteolysis are used by the cells for eliminating proteins, particularly those that are misplaced or dysfunctional.

• Various factors and circumstances influence the rate at which proteins are degraded.