Midterm #2: Protein Targeting Flashcards
1
Q
What are Examples of Post Translational Modifications
A
- Disulfide formation: Cys
- Protein stability, Redox sensing
- Proline/Lysine hydroxylation: Pro, Lys
- Collagen formation & stability
- Glycosylation:
- Asn, Arg : Cell surface proteins
- Ser, Thr, Tyr: Antibodies
- OH-Lys (in collagen): Protein stability
- Phosphorylation: Ser, Thr, Tyr
- Signaling & regulation
- Acetylation: Lys, N-terminus
- Histone protein regulation
- Protein stability & localization
- Proteolytic processing: Backbone
- Hormone production, Pancreatic proteases, Clotting factors
- Glutamate gamma-carboxylation: Glu
- Clotting factors
2
Q
Posttranslational Modification of Collagen
A
- Collagen form triple helix that is the basis of its mecahnical stability
- sequence largely consists of (imperfect) Gly-Pro-Pro repeats.
- Post Translatational Modifications:
- Pro residues are hydroxylated to hydroxyproline, greatly enhancing the stability of the triple helix
- Certain Lys residues are hydroxylated to hydroxylysine and then O- glycosylated. This facilitates protein-protein interactions in the protein’s final (extracellular) environment.
- Inter-strand disulfide bonds between Cys residues trigger formation of the triple helix.
3
Q
- Soluble proteins destined for the cytosol, nucleus and mitochondria are synthesized by
- Proteins bound for the endoplasmic reticulum, Golgi apparatus, lysosomes, plasma membrane or the extracellular environment are synthesized by
A
- ribosomes in the cytosol
- Post-Translational Targeting
- ribosomes bound to the ER
- Co-Translational Targeting
4
Q
Mitochondrial Protein Targeting
A
- ultimate destination of cytosolically synthesized proteins is determined by signal sequences in the nascent polypeptide chain
- Mitochondrial proteins are synthesized on ribosomes in the cytosol as larger “preproteins”, containing an N-terminal mitochondrial matrix targeting sequence that targets the protein to the mitochondria
- mitochondrial entry sequence is a positively charged α-helix, without a well-defined consensus sequence.
- TIM and TOM work in concert to pass preprotein into matrix. Hsp70 to fold into native, proteases to chop into parts, and Hsp60
5
Q
Nuclear Protein Targeting
A
- Nuclear proteins are synthesized in the cytosol.
- Proteins smaller than ~15 kDa can spontaneously enter the nucleus through the nuclear pore complex.
- Larger proteins are targeted by a nuclear localization sequence that includes clusters of positively charged residues.
- NLSs are recognized by importins, which facilitate the transport of larger cargos through the nuclear pore complex
6
Q
Protein Targeting from the ER
A
- ER proteins, Golgi proteins, lysosomal proteins, transmembrane proteins and secreted proteins are synthesized by the rough endoplasmic reticulum (RER).
- proteins are inserted into/through the ER membrane co-translationally and may undergo further processing in the ER and Goldi apparatus. May also be packaged into vessicles
- Signal peptide contained in the N-terminal 15 – 30 residues of the nascent polypeptide signals that a protein is destined for any of these compartments.
7
Q
How proteins get into ER lumen
A
- Signal peptides are recognized by the signal recognition particle (SRP) as they emerge from the ribosome
- When SRP binds to the signal peptide, protein synthesis halts
- SRP-ribosome complex diffuses to the ER surface where it is bound by the SRP receptor in complex with the closed translocon
- GTP hydrolysis by this complex triggers dissociation of the SRP from the ribosome-translocon complex.
- ribosome resumes polypeptide synthesis and the nascent chain passes through the open translocon into the ER lumen.
- signal peptide is cleaved from the growing polypeptide shortly after it enters the ER lumen by a membrane- bound signal peptidase. Polypeptide chains with their signal peptide still attached are known as preproteins.
- When the synthesis of the polypeptide is completed, it is released from both the ribosome and the translocon, and the ribosome dissociates from the RER.
8
Q
Membrane Protein Targeting
A
- ER-resident, Golgi-resident, lysosomal and secretory proteins pass completly thorough the RER membrane into the lumen
- Transmembrane proteins can be transferred directly from the translocon into the ER membrane
- Membrane protein topology can be controlled by a combination of signal peptide, start-transfer and stop-transfer sequences
9
Q
Protein Glycosylation
A
- Many secreted and membrane-associated eukaryotic proteins are glycosylated at Asn, Arg, Ser, Thr or Tyr side chains to generate glycoproteins.
- Glycosylation alters the physical properties of a protein, changing their stability, solubility and physical bulk. In addition, carbohydrate moieties act as recognition signals.
- Oligosaccharides are covalently attached to proteins by either N-glycosidic (Asn, Arg) or O-glycosidic (Ser, Thr, Tyr) bonds by glycosyltransferase enzymes. There are over 100 different glycosyltransferase enzymes
10
Q
N-linked Glycosidation
A
- The sugar residue of N-linked glycoproteins are intially added while the protein is in the ER lumen (core glycosylation)
- Lipid cofactor, dolichol phosphate, is charged with individual monosaccharide subunits to form the core oligosaccharide
11
Q
Name this structure
A
dolichol phosphate
12
Q
- mixture of nucleoside antibiotics that inhibits the first step in N-linked Glycosidation
A
Tunicamycin
13
Q
RXN of N-linked Glycosylation
A
- Complete core oligosaccharide is typically transferred in one step to specific Asn residues of nascent proteins in the ER lumen
- Dolichol pyrophosphate must be dephosphorylated to Dol-P to initate another round of core oligosaccharide synthesis
- Bacitracin inhibits this reaction
- Proteins may be transferred to the Golgi apparatus for further oligosaccharide processing. Certain monosaccharides may be trimmed away by glycosidases. Other sugars (including galactose, fucose, GlnNAc and sialic acid) may be added by specific glycosyltransferases
14
Q
O-linked Glycosylation
A
- O-linked sugars (Ser, Thr, ocassionally Tyr) are added to the proteins while they are in the Golgi apparatus
- sugar residue is transferred from its nucleotide sugar by a corresponding glycosyltransferase
- O-linked oligosaccharides vary in length from one or two sugars in collagen (galactosyl glucose-hydroxylysine) to the chains 100s of disaccharide units in proteoglycans (heparan sulfate, chondroitin sulfate, etc.)
- Lots of glycosylation are proteoglycans (apposed to glycoproteins)
15
Q
Protein Sorting & Trafficking
A
- Mature proteins are sorted in the trans Golgi and sent to their final cellular desitination
- Proteins transit via vesicles that bud off one compartment and fuse with the next.
- This process preserves the orientation of transmembrane proteins
- The carbohydrate portion of a glycoprotein determines the ultimate destination of the protein. (The “default” target is secretion from the cell.)
- Misfolded proteins are exported to the cytosol or lysosome for degradation
16
Q
Bacterial Cell Wall Biosynthesis
A
- Bacterial cell walls consist of a dense network of cross-linked peptidoglycans.
- Peptidoglycan biosynthesis occurs in 3 phases: assembly of precursor in the cytoplasm (bactoprenol vs. dolichol), transport across the inner membrane, and polymerization
-
Bacitracin inhibits the enzyme
responsible for dephosphorylation of bactoprenol-pyrophosphate
(cf. core oligosaccharide biosynthesis). - The β-lactams (e.g., penicillin) inhibit transpeptidase, the enzyme responsible for cross-linking the peptidoglycan chains in the periplasm.
