Midterm 2 Flashcards
(51 cards)
Cell compartmentalization
●membrane-enclosed compartments where biochemical reactions take place
Signal Sequences
●signal peptidases (SPase): remove signals when sorting concludes
●positive, negative, hydrophilic, hydrophobic
●attach to N terminal or C terminal
●import to nucleus (by pores): positive sequence because DNA is negative
●they are released once it arrives where it needs to go
●does not fold into protein
●import into ER is hydrophobic because it crosses membrane
Ribosomes on Endoplasmic Reticulum
●ribosome with mRNA will bind to the ER
●ribosomes bound to ER translate proteins directed to ER
●ribosomes in cytoplasm translate the rest of proteins codified by genome
●Proteins translated by ribosome are translocated to ER
Newly synthesized proteins go into ER
●transmembrane proteins: proteins embedded in ER membrane (cisternae)
●soluble proteins: protein are transferred into lumen of ER
Mechanism of ER signal recognition
●(SRP) signal recognition particle: domain recognizes signal sequence
●mammalian response
●SRP receptor: on ER membrane
●homologous in eukaryotes
●when bound to sequence, causes hinge to fold where it hugs the ribosome
●SRP receptor only recognizes when SRP is bound to ribosome (not fully dependent on sequence)
SRP/Protein translocation into ER
●SRP ensures protein is translated at ER
●Protein translocator interacts with signal sequence and ribosome
●extremely conserved
●Signal sequence on growing peptide-> (recognition) binding of SRP to ribosome causes translation to slow) -> (targeting) interacts with protein translocator -> release ribosome -> recycle SRP
●when signal sequence interacts with protein translocator -> translation continues and translocation begins (protein opens are conformational change) -> translated but released into the ER lumen
●common ribosomes pool
●SRP determines the fate of ribosomes subunits
Protein translocation ER structures
●Plug (short helix) prevents ions to cross membrane: proofreading
●Sec61 complex: protein translocator
●conserved across kingdoms
●Sec62-Sec63: pull polypeptide inside lumen
●BiP proteins (chaperones): avoid sliding back of polypeptide (make sure it stays inside)
●SecA in bacteria: dual function, piston-like protein (SecY, pulls outside cell)
Translocation of Transmembrane proteins (ER)
●recognition of transmembrane domain and signal sequence
●similar for multipass proteins
●signal sequence enters protein translocator -> pulls polypeptide until transmembrane segment
Synthesis of Glycoproteins in ER
●addition of oligosaccharide to lipid
●oligosaccharyl transferase active site in lumen of ER
●Dolichol is lipid anchored to ER membrane and to the precursor of oligosaccharide
●oligosaccharide are synthesized in lumen
●begins in same way as transmembrane -> oligosaccharyl transferase attaches Asp from lipid anchored oligosaccharides anchored in ER membrane
●oligosaccharide has sugar
●glucose is trimmed off
●N-glycosylation
Post-translational modification
●protein glycosylation (add glycan to protein) is most common
ER quality control for protein folding
●Glucosidase (glucose trimming) and glucosyl transferase (glucose addition) have opposite enzymatic activities
●cycles of association and dissociation of glucose
●precursor of oligosaccharide has 3 glucose monomer -> glucose trimming cuts 2 glucose -> calnexin (chaperone that recognize monomer) -> glucosidase (removes glucose) -> make N-linked oligosaccharide -> if folded normally then it leaves the ER
●if not folded properly -> use glucosyl transferase (readds glucose) -> swap uridine diphosphate glucose to UDP and cycle occurs again until protein is folded completely
Misfolded proteins are degraded by proteosome in cytoplasm
●Recognition: chaperon prevents aggregation (bundle w/ other misfolded protein), lectin recognizes oligosaccharide, also disulfide isomerase to break disulfide bond to make it more linear
●targeting: lectin recognizes protein translocator (pulls out protein) complex in membrane of ER, E3 ubiquitin ligase adds ubiquitin to polypeptide (create polyubiquitin complex
●export: AAA-ATPase extracts polypeptide, N-glycanase cleaves oligosaccharide, polypeptide to proteosome (recognize polyubiquitin)
●N-glycanase removed sugar from proteins
ER functions
●Protein translocation
●Protein post-translational modifications (glycosylation)
●phospholipids synthesis
●Ca2+ storage
Phospholipids synthesis
●fatty acids are synthesized in cytoplasm (tail of phospholipids)
●Coenzyme A (CoA) is transferred to a fatty acid
●Acyl Transferase adds glycerol-3-phosphate to two fatty acids -> phosphatidic acid (precursor for phospholipids)
●Fatty acids is bound by fatty acid binding protein and transports them to the ER membrane where acyl-CoA ligase
●Acyl-CoA adds CoA to fatty acid tail at OH then goes to acyl transferase when there are two fatty acid tails close together
●Acyl transferase replaces the CoAs with glycerol 3-phosphate and then further enzymatic modifications occur
Scramblase
●transfers lipid from inner to outer leaflet and vice versa
●needed for polar head to cross hydrophobic layer
●does it randomly
●disruption of phospholipid asymmetry
●scrambles phospholipids when they are synthesized
●no ATP
Flippase
●Transfers lipids using ATP to maintain symmetry
●needed for polar head to cross hydrophobic layer
●maintenance of phospholipid asymmetry
●active
Synthesis of lipids
●glycosphingolipids are synthesized in golgi
●lipids transferred between organelles at contact sites
Cell Nucleus structure
●chromatin
●nucleolus: ribosome assemble
●outer nuclear membrane and inner nuclear membrane: made of phospholipids
●nuclear lamina
●endoplasmic reticulum: directly connected to nucleus
●nuclear pore: where everything passes
Nuclear pore travel
●Import: nuclear proteins (DNA replication proteins, histones, transcription factors)
●shuttling: proteins/RNA
●export: nuclear RNAs (rRNA, mRNA, tRNA, miRNA)
●very conserved
Nuclear Localization Signal (NLS)
●direct proteins to the nucleus
●uses positive signaling sequence
●when mutation occurs, protein remains in cytoplasm (can not transport into nucleus- track with GFP)
ER lumen processes
●proteins fold or undergo post-translational modifications
ER Signal recognition quiz details
●attach to C or N terminal
●recognized by SRP in cytoplasm
●SRP is ribonucleic protein that interact with ribosome and halt transition
●SRP receptor on ER membrane recognize SRP/ribosome complex
Nuclear Pore details
●cytoplasmic filaments: recruit proteins, mRNA, and carriers
●cytoplasmatic, inner and nuclear rings: important for proteins/mRNA to pass through
●nuclear basket
●import receptors are carriers and are enriched in FG sites that interact with FG residues in the ring proteins
Nuclear Import and Export
●part of karyopherin family (move between nucleus and cytoplasm)
●importins bind to cargo (proteins with NLS) and carry them to nucleus
●Exportins bind to cargos with NES and carry them outside nucleus
●Biportins carry cargo in both directions
●process uses energy
