Transport of Large Molecules - Cell Biology PDF

Transport of Large molecules CELL BIOLOGY Don't wait for the opportunity. Create it. L7 Cell Organelles  Transport of Large molecules CELL BIOLOGY Points to be covered in this Lecture Cytosolic and secretory protein Signal peptide and Targeting to RER Membrane...

Transport of Large molecules CELL BIOLOGY Don't wait for the opportunity. Create it. L7 Cell Organelles  Transport of Large molecules CELL BIOLOGY Points to be covered in this Lecture Cytosolic and secretory protein Signal peptide and Targeting to RER Membrane proteins signals Unfolding protein response ER, Golgi, Lysosome  Transport of Large molecules CELL BIOLOGY Transport of macromolecules Endocytosis Exocytosis Phagocytosis Pinocytosis Receptor Regulated Constitutive Mediated endocytosis  Phagocytosis: Cell eating, where cells engulf and internalize solid particles such as bacteria and viruses. Example: Macrophage  Pinocytosis: Cell drinking, cells internalize small dissolved particles, such as ions, sugars, and amino acids, from their extracellular environment.  Receptor mediated endocytosis: cells internalize specific molecules that bind to receptor proteins on their cell surface. Example: Uptake of LDL particles.  Transport of Large molecules CELL BIOLOGY Organelles of Eukaryotic Cells Organalles Wthout Single Double Membrane Membrane Membrane Ribosome, Centriole ER, Golgi, Lysosome, Mitochondria, Chloroplast, Vacoule, Peroxisome Nucleus Transport of Large molecules CELL BIOLOGY Cytosolic and Secretory Protein Proteins Cytosolic Secretory Ribosome, cytosol, nuleus, ER, Golgi, Lysosome, Vacoule, mitochondria, chloroplast, plasma Membrane, outside Peroxisome cell  Transport of Large molecules CELL BIOLOGY Cytosolic Secretory protein protein Synthesis starts Synthesis starts on free and complete on RER membrane ribosome free ribosome But completed on RER membrane Transport of Large molecules CELL BIOLOGY Transport of Large molecules CELL BIOLOGY  Transport of Large molecules CELL BIOLOGY Cotranslational targeting of secretory proteins to the ER  Transport of Large molecules CELL BIOLOGY Hydrophobic signal s Recognize by Signal Recognition Particle (SRP) Ribonucleoprotein - 300 nt –RNA + 54 KD protein SRP SRP interacts with Ribosome → Translation Pause SRP interacts with – 1. signal peptide 2. Large Subunit of Ribosome 3. SRP Receptor present on RER membrane  Transport of Large molecules CELL BIOLOGY Step 1: As the signal sequence emerges from the ribosome, it is recognized and bound by the signal recognition particle (SRP). Step 2: The SRP escorts the complex to the ER membrane where it binds to the SRP receptor. Step 3: The SRP is released, the ribosome binds to the translocon, and insertion of the signal sequence opens the translocon. Step 4: Translation resumes and the signal sequence is cleaved by signal peptidase. Step 5: Continued translation drives translocation of the growing polypeptide chain across the membrane. Step 6: The completed polypeptide chain is released within the ER lumen.  Transport of Large molecules CELL BIOLOGY Signal peptidase RER Lumen - Cleave signal Peptide & Generate New N- Terminus Chaperon present in RER Lumen - Bip Belongs to hsp70 family Prevent pre-mature folding of Protein Hiding the Hydrophobic amino acid. in their hydrophobic pocket Assist Protein folding RER Lumen Transport of Large molecules CELL BIOLOGY  Transport of Large molecules CELL BIOLOGY N-linked glycosylation Important for Protein Folding Glycosylation provide stability to the protein Proteins are glycosylated within the ER by the addition of a 14-sugar oligosaccharide to an acceptor asparargine (Asn) residue. Three glucose residues are removed while the protein is still within the ER Transport of Large molecules CELL BIOLOGY  Transport of Large molecules CELL BIOLOGY The oligosaccharide is synthesized on a lipid carrier anchored in the ER membrane. It is then transferred as a unit to acceptor asparagine residues in the consensus sequence Asn-X-Ser/Thr Glycosylation helps to prevent protein aggregation in the ER and provides signals that promote protein folding and subsequent sorting in the secretory pathway. Transport of Large molecules CELL BIOLOGY  Transport of Large molecules CELL BIOLOGY Protein Di-Sulphide isomerase- Helps in Di-sulphide Bond formation- Rearrangement of Di-sulphide Bond Oxidative Environment present in RER Lumen → Di-sulphide Bond can formed Cytosolic environment – di sulphide Bond can not form in cytosolic protein Except - Keratinocytes Some how maintain oxidative environment in cytosol Kiratin is cytosolic protein but contain s-s Bond  Transport of Large molecules CELL BIOLOGY Prolyl cis - trans isomarase Pro-Pro ( cis trans )  Transport of Large molecules CELL BIOLOGY Quality Control of Folded Protein → Properly folded Proteins. leaves RER Lumen and enters in Golgi Chaparons(Вір) - Unflolding misfolded protein =Transport to the cytosol (ER mediated degradation).Refold→ Correct Folding -- Packed into vesicle ----send into vesicle Cal reticulin Calnexiin Glucosidase Mannosidase UDP Glycosyl Transferase  Transport of Large molecules CELL BIOLOGY Glycoprotein folding As the glycoprotein exits the translocon, chaperones bind and assist in folding. If the protein is correctly folded it proceeds to exit the ER. However, if too many hydrophobic regions are exposed, indicating improper folding, the protein is targeted back to the cytosol through a ubiquitin ligase complex in the ER membrane. The protein is ubiquitylated at the cytosolic side of this complex and degraded in the proteasome  Transport of Large molecules CELL BIOLOGY If there is mutation in protein –(single copy mutation of protein) Correct folding does not occurs ER Associated Degradation for mis-folded protein If RNA is mutated – unfolded protein Response (UPR)  Transport of Large molecules CELL BIOLOGY UPR Mutated RNA Degradation global translational inhibition ( PER-K add P on eIF2 ----| Translation Chaperon expression  Transport of Large molecules CELL BIOLOGY The export and degradation of misfolded ER proteins.  Transport of Large molecules CELL BIOLOGY Misfolded soluble proteins in the ER lumen are recognized and targeted to a translocator complex in the ER membrane. They first interact in the ER lumen with chaperones, disulfide isomerases, and lectins. They are then exported into the cytosol through the translocator. In the cytosol, they are ubiquitylated, deglycosylated, and degraded in proteasomes. Misfolded membrane proteins follow a similar pathway but  Transport of Large molecules CELL BIOLOGY The unfolded protein response (A) By three parallel intracellular signaling pathways, the accumulation of misfolded proteins in the ER lumen signals to the nucleus to activate the transcription of genes that encode proteins that help the cell cope with misfolded proteins in the ER. Transport of Large molecules CELL BIOLOGY  Transport of Large molecules CELL BIOLOGY Insertion of a membrane protein with a cleavable signal sequence  Transport of Large molecules CELL BIOLOGY Insertion of a membrane protein with a cleavable signal sequence The signal sequence is cleaved as the polypeptide chain crosses the membrane, so the amino (N) terminus of the polypeptide chain is exposed in the ER lumen. However, translocation of the polypeptide chain across the membrane is halted when the translocon recognizes a transmembrane sequence. This allows the protein to exit the translocon laterally and become anchored in the ER membrane. Continued translation results in a membrane-spanning protein with its carboxy (C) terminus on the cytosolic side  Transport of Large molecules CELL BIOLOGY Insertion of membrane proteins via internal transmembrane sequences I The transmembrane sequence directs insertion of the polypeptide such that its amino (N) terminus is exposed on the cytosolic side. The transmembrane sequence exits the translocon to anchor the protein in the lipid bilayer and the remainder of the polypeptide chain is translocated into the ER as translation proceeds  Transport of Large molecules CELL BIOLOGY Other internal transmembrane sequences are oriented to direct the transfer of the amino-terminal portion of the polypeptide across the membrane. Continued translation results in a protein that spans the ER membrane with its amino terminus in the lumen and its carboxy (C) terminus in the cytosol.  Transport of Large molecules CELL BIOLOGY Insertion of a protein that spans the membrane multiple times  Transport of Large molecules CELL BIOLOGY Topology of the secretory pathway  Transport of Large molecules CELL BIOLOGY A “road-map” of the secretory and endocytic pathways. Transport of Large molecules CELL BIOLOGY Transport of Large molecules CELL BIOLOGY Transport of Large molecules CELL BIOLOGY Transport of Large molecules CELL BIOLOGY  Transport of Large molecules CELL BIOLOGY Endoplasmic reticulum Transport of Large molecules CELL BIOLOGY Transport of Large molecules CELL BIOLOGY  Transport of Large molecules CELL BIOLOGY Rough ER Structure Cisternae (well developed with ribosome), tubules, vesicles Function Synthesis of secretory protein Protein folding and Quality Control Disulphide bond formation N-linked glycosylation (occurs on R-chain of Asn of protein) Transport of Large molecules CELL BIOLOGY  Transport of Large molecules CELL BIOLOGY Smooth ER Structure Cisternae, tubules (well developed) and vesicles Function Lipid biosynthesis (Phopholipid, Tri Acyl Glycerol) Steriod biosynthesis (Cholesterol, Steroid hormones) Detoxification of drugs (Cytochrome P450- Monoxygenase) Storage of Ca2+ (sarcoplasmic reticulum) Transport of Large molecules CELL BIOLOGY Transport of Large molecules CELL BIOLOGY Transport of Large molecules CELL BIOLOGY Transport of Large molecules CELL BIOLOGY  Transport of Large molecules CELL BIOLOGY Lysosome Structure Single membrane bound spherical vesicle like structure Lumen has acidic pH (4.5) in lumen due to V-type of pump Marker membrane protein : LAMP-1 Have variiety of acid hydrolases Function Heterophagy: Digest extra cellular content like bacteria, virus. Autophagy: Degrade cells own content like non-functional mitochondria. Marker of autophagy is LC3 protein. Termed as suicidal bag of cell Transport of Large molecules CELL BIOLOGY  Transport of Large molecules CELL BIOLOGY Golgi Complex  Golgi complex consist of cisternae, tubules and vesicles.  It has definite polarity – cis golgi toward RER or nucleus while trans golgi network toward plasma membrane Transport of Large molecules CELL BIOLOGY Transport of Large molecules CELL BIOLOGY Transport of Large molecules CELL BIOLOGY Transport of Large molecules CELL BIOLOGY  Transport of Large molecules CELL BIOLOGY A “road-map” of the secretory and endocytic pathways.  Transport of Large molecules CELL BIOLOGY Golgi Complex Important function of Golgi complex are  Protein sorting and protein secretion  O-linked glycosylation on R-chain of serine or threonine  Addition of mannose 6- phosphate for lysosomal proteins  Carbohydrate biosynthesis – pectin and hemi-cellulose  Synthesis of sphingolipids  Formation of endosome, lysosome, vacuole.  Acrosome formation in sperm  Transport of Large molecules CELL BIOLOGY Lysosome Structure Single membrane bound spherical vesicle like structure Lumen has acidic pH (4.5) in lumen due to V-type of pump Marker membrane protein : LAMP-1 Have variiety of acid hydrolases Function Heterophagy: Digest extra cellular content like bacteria, virus. Autophagy: Degrade cells own content like non-functional mitochondria. Marker of autophagy is LC3 protein. Termed as suicidal bag of cell Transport of Large molecules CELL BIOLOGY Transport of Large molecules CELL BIOLOGY Transport of Large molecules CELL BIOLOGY  Transport of Large molecules CELL BIOLOGY Lysosomal Storage Diseases Name of Disorder Mutant Enzyme Gaucher’s disease Glucocerebrosidase Tay-Sach’s Disease GMT-2 Gangliosidase or β-N-acetylhexosaminidase, Pompe II Disease Alpha-1,4-Glucosidase (glycogen accumulates) Niemann-Pick Disease Spingomyelinase Von-Gerke Disease Alpha-1,6-Glucosidase (glycogen accumulates) Hurler Syndrome α-L-iduronidase (glycosaminoglycans accumulate) Metachromatic Arylsulfatase (cerebroside-sulfatase accumulate) leukodystrophy Transport of Large molecules CELL BIOLOGY  X-linked adrenoleukodystrophy, Zellweger syndrome (ZS) and Refsum Transport disease are diseases of Large related with peroxisomes. molecules CELL BIOLOGY Functions of Vacoule It maintains cell turgor and cell shape Keeps the chloroplasts closer to light due to push by vacoule. Store inorganic ions (like K+ and Cl-), salts (such as calcium), and other substances, including toxic byproducts which may interfere with metabolism in cytosol. Accumulates toxins which help to protect from herbivores. Stores the pigments in flowers and fruits. It contains hydrolytic enzymes, help in recycling of cellular constituents especially during senescence. Regulates cytosolic pH with help of ATP-dependent V-type of proton pumps  Transport of Large molecules CELL BIOLOGY Peroxisomes and Vacuole Functions of Peroxisomes Oxidation reactions of organic compounds Catabolism of very long chain fatty acids, branched chain fatty acids, D-amino acids and polyamines. Biosynthesis of plasmalogens and ether phospholipids Metabolism of ureides and reactive oxygen species (H2O2) Beta oxidation of fatty acid and glyoxylate cycle in plants Marker enzyme is catalase X-linked adrenoleukodystrophy, Zellweger syndrome (ZS) and Refsum disease are diseases related with peroxisomes. Functions of Vacoule It maintains cell turgor and cell shape Transport of Large molecules CELL BIOLOGY L1: DNAofReplication Transport Large molecules CELL BIOLOGY Transport of Large molecules CELL BIOLOGY