Protein Targeting Flashcards
Protein targeting
Two sites of protein synthesis (cytosol and rough ER)
All proteins start their synthesis on ribosomes free in the cytosol
Two types of proteins are synthesized in the ER:
- transmembrane proteins (LDL receptor)
- Water soluble proteins
Ribosomes in the cytsol will send proteins to the mito, peroxisome, or the nucleus
Ribosomes in the ER will send proteins down the secretory path (golgi, lysosomes, cell surface, and secretory vesicles)
Cytosolic vs ER bound Ribosomes
they are the same
signal recognition particle (SRP) cycle
SRP is a ribonucleoprotein composed of a single 7 SL RNA molecule of 300 nucleotides plus 6 different polypeptide chains, it cycles between the ER membrane and the cytosol, it binds to the signal sequence and the ribosome to function in signal recognition and elongation arrest of the poly peptide chain
Signal sequence is the tag/zipcode:
no consensus sequence (primary sequence differs between signal sequences), adopt a similar threeD structure recognized by the SRP usually occurs at the amino terminus of a protein (if it is at the amino terminus it is cleave co translationally by signal peptidase). (Tripartate domain sequence (aka has a hydrophillic amino terminal domain that has a positive chain, and a hydrophobic core domain(7 residues in an alpha helix, and a carboxyl terminal domain)
The SRP-receptor is expressed exclusively in the ER, its an integral membrane protein with the SRP binding site exposed to the cytosol. It binds to the SRP in the new SRP/protein/Ribosome complex and will send the complex to the ER membrane (GTP REGULATES this step)
Translocation process: the import of a water soluble peptide into the lumen of the ER: Requires ATP hydrolysis, and occurs thru an aqueous pore/channel called a translocon. Translocation and protein synthesis is coupled since the unfolded peptide is whats preferentially important thru the translocon. Translocated peptides are folded into native state in the ER lumen with the assistance of chaperones.
Topology and stop transfer sequences
Stop transfer sequences are hydrophobic, alpha helical sequences that anchor the protein into the membrane. Proteins that span the membrane many times have many stop transfer sequences (GLUTs and G protein coupled receptors)
Targeting to plasma membrane and secretory granules
Proteins (soluble or membrane bound) are carried out to the cell surface UNLESS they have a signal that direct them elsewhere
Constituitive pathway- common to all cells soluble proteins are continously secreted without intracellular storage
regulated secretory pathway: present in certain cells (exocrine, endocrine, neurons)- the secretory proteins are stored in storage organelles (secretory granules) and are released upon stimulation
Lysosomes
The Recycling center of the cell
Lysosomes has 60 different hydrolytic enzymes to digest intracellular macromolecules
Degraded macromolecules are recycled and used in pathways for energy production (TCA cycle) or in biosynthetic pathway (protein synthesis)
All hydrolytic enzymes are acid hydrolases active at ph 5(lysosomal ph)
V-type ATPases are responsible for acidifying intracellular compartments
Functions of lysosome: disposal of abnormal proteins, downregulation of cell surface signaling receptos, release of endocytosed nutrients (cholesterol from LDL), phagocytosis, cell survival (autophagy ie self eating)
3 pathways to lysosome: phagocytosis, endocytosis, autophagy
neurodegenerative diseases and autophagy
protein aggregates and damagend organelles accumulate within specific neurons of pateients: alzheimers, parkinsons, huntingtons, als
How to build a lysosome
Manose 6-phosphate receptors recognize the zipcode on the mannose 6 phosphate tagged lysosomal hydrolytic enzymes from the golgi to the pre lysosome
Lysosomal enzymes (acid hydrolyases) are synthesized in the ER and transported thru the golgi, the proteins carry a signal sequence to target them to ER for their synthesis. Soluble acid hydrolases are tagged with mannose -6 phosphate on N-linked glycans to direct them to lysosome by mannose 6 phosphate receptors
Lysosomal enzymes are glycoproteins with n linked glycans
Targeting of acid hydrolases to the lysosome: glycosylation and glycosidases modify glycans, location of Glc-NAc phosphotransferases and uncovering enzyme
Lysosomal storage diseases treatment
loss of any one lysosomal storage enzyme leads to disease, results in accumulation of undegraded material
FDA approved therapies exist for only a few diseaes, enzyme replacement therapy involves IV injection of the missing enzyme every 2 weeks
Treatment depends upon receptor mediated endocytosis of mannose 6 phosphate tagged enzyme with the mannose 6 phosphate receptor
Mucolipidosis 2 I cell disease
Pompe disease
lysosomal storage defect is in the generation of the mannose 6 phosphate tag
symptoms at birth psychomotor retardation skeletal abnormalities, hepatosplenomegaly, lots of shit
Glc-NAc- phosphotransferase deficiency
Pompe disease: undegraded glycogen (deficient alpha-glucosidase) death before 2 years cardiomyopathy and respiratory issues
Molecular basis of vesicular transport
vesicular transport between organelles involves:
- coat protein covered vesicle that buds from the donor organelle
- coat proteins are released from the vesicle
- the uncoated vesicle binds to a specific target compartment
- fusion of the uncoated vesicle to the target membrane
SNARE proteins provide the specificity in the fusion of all vesicles with target membranes. SNARE is a family of integral membrane proteins. Each vesicle has its own v-SNARE which pairs with the cognate t-SNARE found only on the specific target membrane. This unique matching ensures specificity of the fusion event
SNARES act as a winch to bring membranes together (fusion
vesicle move along the tracts (ie micro tubles)
Tetanus and botox: caused by extremely potent neurotoxin that inhibits the release of NT from presynaptic membranes
Botox prevents Ach release-> flacid paralysis
Tetanus-> blocks release of GABA (inhibitory NT) and results in spastic paralysis (lock jaw)
tetanus and botox are peptidases cleave SNARES essential for synaptic vesicle fusion to the plasma membrane
Receptors-mediated endocytosis
An efficient pathway for taking up specific macromolecules from the extracellular fluid, process invovles a selevtive concentrating mechanims (increases efficiency of internalized so that you dont take up large amounts of fluid)
The receptors can recycled and ligand degraded, ligand and receptor can be degraded, both can be recycled, both are transported
LDL-> gets degraded but the receptor is recycled
transferrin-> both are recycled
Clathrin
major protein component of coated pits/vesicles
triskeleton 3 heavy and 3 light chains
-2% PM
Recycled extensively
Transferrin receptor and iron uptake
Transferrin transports iron in liver cells-> lysosome where the pH releases the iron and (apotransferrin replaces the iron ) transferrin receptor/apotransferrin released extracellular
familial hypercholesterolemia
- elevated levels in concentration of LDL in plasma
- deposition of LDL derived cholesterol in skin, tendons and arteries
- inheritance autosomal defect in LDL receptor
Heterozygotes 1/500
homozygotes 1/milillion (treatment lowere LDL)
Transport clustering and recycling
