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.

Explore the intricate processes involved in protein distribution and transport within eukaryotic cells. This quiz covers mRNA translation, protein synthesis, and the roles of cellular organelles such as the endoplasmic reticulum and Golgi apparatus. Test your understanding of protein regulation and transport mechanisms.