13 - Intracellular membrane traffic Flashcards
Different coated vesicles are used for different transport steps
clathrin coated vesicles transport material from plasma membrane and endosomal/Golgi membranes (back and forth)
COPI coated vesicles bud from Golgi compartments
COPII coated vesicles bud from ER
Phosphoinositides
Phosphoionositides (PIPs) mark organelles and membrane domains by recruiting proteins that bind the PIPs to matching PIP domains. The PIP-binding proteins then help regulate vesucle formation and other steps in the vescile traffic.
The endocytic pathway
clathrin-covered (triskelion outer coat, adaptor proteins inner coat)
DYNAMIN
Inner coat mediates cargo selection, outer coat deforms the membrane to generate vesicle.
cargo binds to cargo receptors (bound to adaptor proteins), which recruits clathrin subunits. This causes the formation of a small bud. membrane-binding proteins that have BAR domains help impose their shape on the membrane via electrostatic interactions with the lipid head groups, allowing the membrane to form a vesicle.
soluble cytoplasmic proteins like dynamin are present at the neck of the bud/vesicle. Two non-cytosolic leaflets of the membrane are brought into close proximity and fused. Tgis is done by dynamin and other proteins.
Once released from the membrane, the vesicle loses its clathrin coat, as the binding of the adaptor proteins is weakened. Combination of PIP phosphatase located in the vesicle membrane and hsp70. a hsp70 chaperone protein also helps the uncoating by ATP hydrolysis.
Coat-recruitment GTPases, like ARF proteins and Sar1 protein also involved in coat disassembly. GTP hydrolysis causes conformational change ecposint hydrophobic tail, and thus release of coat.
As endosomes mature, patches of their membrane invaginate into the endosome lumen to form intralumenal vesicles, and the maturing endosomes can be called multivesicular bodies. Ubiquitylated membrane protein are sorted into domains on the endosomal membrane by ESCRT proteins (which bind ubiquityl), which invaginate and pinch oss (sequentration) to form intralumenal vesicles. The ubiquityl marker is removed and returned to cytosol for reuse. Proteases and lipases in lysosomes will ingest these intralumenal vesicles once the endosome has fused with a lysosome.
COPII transport vesicles
dependent on exit signals int he cargo proteins. coat is only disassembled upon arival at target membrane through phosphorylation of coat proteins
Sar1-GDP (inactive, but coat-recruiting GTPase) binds to a Sar1 GEF (activator) in the ER membrnae, causing Sar1 to release GDP and exchange it with a GTP. This causes a conformational change in Sar1 that initiates membrane bending.
GTP bound Sar1 also binds two COPII adaptor coat proteins (Sec23 and Sec24) which form the inner coat. A complex of two other COPII coat proteins (Sec13 and 31= forms the outer shell of the coat. Membrane-bound, active Sar1-GTP recruits COPII adaptor proteins to the membrane. They select certain transmembrane proteins and cause the membrane to deform. the adaptor proteins help recruit the outer coat proteins which help form a bud. A subsequent membrane fusion event pinches off the coated vesicles
COPI transport
KDEL (Lys-Asp-Glu-Leu) = retrieval signal for retrograde ttransport of soluble ER proteins (like BiP). Recognized by KDEL receptor protein, binds proteins with KDEL and integrates it into COPI vesicle (preassembled). Disassembly of COPI involves ARF GTPase
what type of coat?
clathrin = plasma membrane and back and forth to Golgi and endosomal membranes
COP II retrieves proteins from ER
COP I bud from Golgi
Targeting of the vesicle - where to?
Rab and SNARE proteins.
Rab: direct vesicles to specific spots on target membranes. Mode of action similar to Sar1, they are inactive bound to GDP in cytosol, and active in membrane bound form (anchored when GDP -> GTP). This recruits more active Rab-GEFs (to make more GDP->GTP), and Rab to the same site as well. Active Rab also activates a PI 3-kinase, which locally converts PI to PI(3)P, which binds some of the Rab effectors and stabilizes their local membrane attachment. This is positive feedback.
SNAREs: v- and t- snares (vesicle and target), help mediate the fusion of the lipid bilayers.
After the Tab proteins have established the connection between the two membranes to fuse, SNAREs take over. SNARE proteins on the two membranes interact in paris, docking the vesicle to the target membrane and catalyzing the fusion. During fusion, Rab hydrolyses its GTP and leaves as a soluble protein.
interactions between v- and t-SNAREs makes a trans-SNARE. trans-SNARES catalyze membrane fusion by using the energy that is freed when the interacting helixes wrap around each other to pull the membranes together, and squeezing out water molecules (blocks the fusion) from the fusion site. ¨
The protein NSF cycles between membranes and cytosol and catalyzes dissasembly of SNARE complexes (unravel the helices of v- and t-)
Transport from ER through Golgi
Golgi = major compartment for carb synthesis, and sorting and dispathcing station for products of the ER.
Proteins leave ER in COPII vesicles. proteins without the exit signals can also enter these transport vesicles to leave, including ER-resident proteins (leak out).
These vesicles can fuse with one another in the cytosol after leaving ER - heterotypic or homotypic (based on membrane origin). These clusters are called vecisular tubular clusters. As soon as they form, they begin to bud off transport vesicles of their own (not COPII, but CPOI coated). The COPI-coated vesicles function as a retrieval pathway, carrying back ER resident proteins that have escaped, and proteins like cargo receptors and SNAREs). This is the retrieval/retrograde transport. The retrieval of proteins continues in the Golgi.
The retrieval pathway uses sorting signals (KDEL receptor binds to a protein and leads it into a COPI-coated retrograde transport vesicle.
The Golgi Apparatus
cis face = entry face (cis golgi network, CGN), trans face = exit face (TGN).
Both networks are important for protein sorting, through CGN they can move onward in Golgi or return to ER, proteins exiting from TGN move to endosomes, secretory vesicles, or cell surface.
Oligosaccharide chains are processed in the Golgi:
- protein arrives from ER
- Sorting: phosphorylation of oligosaccharides on lysosomal proteins
- removal of mannose
- addition of N-acetylglucoseamine
- addition of galactose and sialic acid
- sulfation of tyrosines and carbohydrates
- sorting (for lysosome, plasma membrane or secretory vesicle.
Oligosaccharide processing in the ER and Golgi (whole process)
- in ER lumen, processing begins with removal of glucoses from the oligosaccharide initially transferred to the protein. A mannosidase in the ER membrane removes a specific mannose.
- in Golgi (rest): Golgi mannosidase removes three more mannoses
- N-acetylglucosamine transferase adds an N-acetylglucosamine
- mannosidase removes 2 more mannoses. This yields the final core of three mannoses that is present in a complex oligosaccharide
- Additional N-acetylglucosamines, galactoses and sialic acids are added. These final steps in the synthesis of a complex oligosaccharide occur in the cisternal compartments of Golgi.
Transport through the Golgi
two models; cisternal maturation and vesicla transport
Cisterna lmaturation model: each golgi cisterna matures as it migrates outward through the stack. At each stage, the Golgi resident proteins that are carrieed forward in a maturing cisterna are moved backward to an earlier compartment in COPI-coated vesicles. When a newly formed cisterna moves to a medial position, “leftover” cis Golgi enxymes would be extracted and transported reterogradedly to a new cis cisterna behind.
Vesicle transport model: Golgi cisternae are static compartments, which contain a characteristic complement of resident enzymes. The passing of molecules from cis to trans through the Golgi is accomplished by forward-mocing transprot vesicles, which bud from one cisterna and fuse with the next in a cis-to-trans-direction
What is the purpose of glycosylation?
N-linked oligosaccharides make the protein more soluble, thereby preventing aggregation and promoting proper folding, and they also establish a “glyco-code” that marks the progression of protein folding and mediates the binding of the protein to chaperones.
Cellular trsansport network
Endocytic pathway: Plasma membrane to early eendosome to late endosome to lysosome
Secretory pathway: ER membrane to Golgi to secretory vesicle OR early endosome (and late + lysosome) OR to late endosome (and lysosome)
Retrieval pathways: endosome (late or early) to plasma membrane OR Golgi to ER
Lysosomes
pH ca 5, maintained by ATPases pumping protons inside it
contains nucleases, proteases, glycosidases, phosphatases, sulfataases and phospholipases that can cleave different compounds. These enzymes aredelivered from ER via Golgi
Lysosome maturation
Late endosome merges with a lysosome to make an endolysosome (endosomes usually fuse with each other). when the contents have been lysed the endolysosome is called a lysosome again.
