Unit 6 Flashcards
(28 cards)
Signal sequences of proteins
- Used to identify where they’re headed
Sufficient vs necessary signal sequences of proteins
- Sufficient: All a protein needs to be imported in an organelle
- Necessary: A protein needs this to be imported in an organelle
- All sufficient amino acid sequences are necessary
3 types of protein sorting
1.) Recognition of the signal sequence by a shuttling cyotcylic receptor
2.) Targeting to the outer surface of the organelle membrane
3.) Import of the targeted protein into the membrane or transport of the protein across the membrane
3 main mechanisms to import proteins into a membrane-enclosed organelle
1.) Transport through nuclear pores
a.) Transports specific proteins
b.) Proteins remain folded during transport
2.) Transport across membranes
a.) ER, mitochondria, chloroplasts, peroxisomes
b.) Requires protein translocators
c.) Proteins are unfolded in order to cross the membrane
3.) Transport by vesicles
a.) From ER onward & through endomembrane system
b.) Transport vesicles collect cargo protein & pinch off from membrane
c.) Deliver cargo by fusing with another compartment
d.) Proteins remain folded during transport
Mitochondrial import
1.) Precursor proteins are kept in an unfolded state by the action of the cytosolic chaperone Hsc70. This requires energy in the form of ATP hydrolysis
2.) Matrix-targeting sequence then interacts with a receptor in the outer mitochondrial membrane called TOM20 or TOM22
3.) Receptor transfers the protein to the general import pore of the outer membrane composed of the protein TOM40
4.) At contact sites between the inner & outer membranes, the protein passes through the import pore of the inner membrane composed of the proteins TIM23 & TIM17
5.) Matrix Hsc70 binds to TIM44. ATP hydrolysis by this complex helps power translocation of the protein into the matrix
6.) As the matrix-targeting sequence emerges in the matrix, it is cleared by a matrix protease
7.) The protein can then fold into its final conformation, often (but not always) assisted by matrix chaperones
2 separate populations of ribosomes in cytosol
1.) Membrane-bound ribosomes are attached to the cytosolic surface of the ER membrane & are synthesizing proteins that are translocated into the ER
2.) Free ribosomes are unattached to any membrane & are synthesizing all of the other proteins
Co-translational translocation
- Done by the ER, the protein is translocated to ER from free ribosomes mid-translation
ER Import
1, 2.) The emerging polypeptide with its ER signal sequence exposed is engaged by a complex of six proteins and an associated RNA molecule called the signal recognition particle (SRP). This binding halts translocation and delivers the ribosome / polypeptide to the ER
3.) SRP delivers the ribosome / polypeptide to SRP receptor. This interaction is enhanced by the binding of GTR to both SRP & its receptor
4.) The ribosome / polypeptide is then transferred to the translocon, inducing it to open & receive the polypeptide which enters as a loop. Hydrolysis of GTP by SRP & its receptor free these factors for another round of import
5, 6.) Translation then resumes & the signal sequence is cleaved by a membrane-bound protease called signal peptidase. Following this digestion, the rest of the protein is synthesized & enters the lumen of the ER
7, 8.) Following completion of translation, ribosome is released causing the translocon to close. The newly-synthesized protein then folds.
6 main types of membrane-anchored proteins
1.) Type I: Single pass, cleaved signal sequence at the N-terminus, uses SRP-SRP receptor to get to ER membrane, N(out)-C(in)
2.) Type II: Single pass, no cleavable signal sequence, uses SRP-SRP receptor to get to ER membrane N(in)-C(out)
3.) Type III: Same as type II but N(out)-C(in)
4.) Tail-anchored: Single pass, no cleavable signal sequence, uses SRP-SRP receptor for insertion of the first membrane-spanning domain but not subsequent ones, IV-A are N(in)-C(out), IV-B are N(out)-C(in)
6.) GPI-anchored): Entire protein is lumenal (out), cleaved signal-sequence at the N-terminus, uses SRP-SRP receptor to get to ER membrane, anchored at C-terminus to membrane and then transferred to GPI anchor
Hydropathic plots: Help determine the type of membrane protein
- The more hydrophobic the amino acid, the more positive the hydropathic index
- The more hydrophillic the amino acid, the more negative the hydropathic index
- The hydropathic index for groups of 20 amino acids are calculated and plotted against the protein sequence
Besides proteins that reside in ER, ER is the starting point for:
1.) Soluble proteins that will be secreted from cell
2.) Soluble proteins that are destined for Golgi, lysosome, or endosomes
3.) Membrane proteins that will embed in the Golgi, lysosome, endosomes or plasma membrane
4 principle modifications that occur in ER
1.) disulfide bond formation
a.) Stabilise protein structure
b.) Covalent bond either on the same or different proteins
2.) Glycosylation
3.) Folding of polypeptide chains and assembly of multisubunit complexes
4.) Protealytic cleavage of amino-terminal sequences
Roles of glycosylation
1.) Promotes folding of a protein
2.) Provides some stability to the proteins
3.) Promote cell-cell adhesion on plasma membrane proteins
4.) Act as a transport signal
3 main classes of vesicle coats
1.) Clathrin: Mediates transport vesicle formation of the trans-golgi and of the plasma membrane
2.) COP I: Mediates transport from the cis-Golgi to the ER of between various of Golgi cisternae
3.) COP II: Mediates transport from the ER to the cis-Golgi
3 functions of protein coat on the cystolic surface of budding vesicles
1.) Shapes the donor membrane into a bud
2.) Helps to capture cargo proteins into budding vesicles
3.) Coat formation & other steps in vesicle formation require small GTP binding protins called Rab proteins. Cycle between active (GTP-bound) and inactive (GDP-bound) forms
Steps involved in COP II coat formation
1, 2.) Rab protein Sar1 is activated by its GEF. It then inserts into the membrane & begins to curve the membrane
3.) Activated Sar1 recruits the inner portion of COP II coat made up of the proteins Sec23 & Sec24. These proteins further bend the membrane. Sec24 acts as a cargo receptor for membrane proteins
4.) Sec23 & Sec24 recruit the outer layer of the COP II coat made up of the proteins Sec13 & Sec31
Common features of fusion of transport vesicles to their target membrane
1.) Vesicle coats must be completely / mostly removed from vesicle
2.) Vesicle must be specifically recognised by current destination membrane
3.) The vesicle & target membrane must fuse & mix to deliver the contents of the vesicle to the target organelle
Vesicle coat disassembly
- Formation of the GPI coat at golgi requires
a.) Arf I (a Rab protein) activation
b.) COP I complex composed of 7 subunits recuited en bloc (as one unit)
Main steps in vesicle-mediated transport, after vesicle formation
1.) Tethering-mediated by Rabs & their effectors, tethering factors & SNAREs
2.) Docking-mediated by SNARE pairing
3.) Fusion-driven by SNARE “zippering”
Several classes of tethers
1.) Multiprotein tethering complexes - composed of up to 10 proteins, localised to distinct organelles
2.) Coiled - coil proteins (long a-helical proteins) that project great distances from target membrane
- Docking-stranger interaction between the vesicle & the target membrane. Occurs over a short distance
Membrane fusion
- Fusion happens in 3 stages:
1.) Outer leaflet mixing between the vesicle & target membranes to produce a hemifusion intermediate
2.) Expansion of the hemifusion intermediate provides a surface for the inner leaflets to fuse
3.) Fusion of inner leaflets allow access of the soluble material in the vesicle & target membrane to mix
2 models describing how a protein travels to other Golgi cisternae after arriving in cis-Golgi
1.) Vesicle transport model: Golgi cisternae are stable, soluble compartments that receive & transport cargo in anterograde - directed (ER-Golgi-PM) vesicles
2.) Cisternal maturation model: Secretory cargo is static & passivley matures as Golgi enzymes from later compartments travel in retrograde-directed vesicles
Different ways of Golgi modifying proteins
1.) Addition of galactose & other carbohydrates takes place in trans Golgi
2.) Addition of GlcNAc, fucose & additional mannose trimming takes place in the medial Golgi
3.) Mannose trimming takes place in cis Golgi
2 main types of endocytosis
1.) Bulk-phase endocytosis: Non-selective, can be clathrin-dependent or clathrin-independent
2.) Receptor-mediated endocytosis: Selective, clathrin-dependent, initiated by the binding of a ligand to its receptor
