6 - intracellular compartments & protein transport

exocytosis

vesicle fuses with plasma membrane releasing its contents into the cell’s surroundings

endocytosis

extracellular materials are captured by vesicles that bud inward from the plasma membrane and are carried into the cell

maintenance of orientation

cytosolic side always faces cytosol, noncytosolic side always faces the outside of the cell

outward secretory pathway

protein molecules are transported from the ER, through the golgi, to the plasma membrane, to the lysosomes

inward endocytic pathway

extracellular molecules are ingested in vesicles derived from the plasma membrane and are delivered to early endosomes and to lysosomes via late endosomes

vesicle buddying is driven by…

assembly of a protein coat

clathrin molecules

form basketlike cages that help shape membranes into vesicles

clathrin coated vesicle transport

• cargo receptors with their bound cargo molecules are captured by adaptins which also bind clathrin molecules to the cytosolic surface of the budding vesicle

• dynamin proteins assemble around the neck of budding vesicles and hydrolyze their bound GTP to pinch off the vesicle

• after budding is complete, coat proteins are removed and the naked vesicle can fuse with its target membrane

mutant dynamin protein in flies

clathrin coated pits assemble and dynamin is recruited around the neck of the budding vesicles but fail to pinch them off —> paralysis bc clathrin mediated endocytosis stops, preventing the recycling of vesicles needed to release neurotransmitters

vesicle docking depends on…

tethers and SNAREs

tethering protein binds to a Rab protein on the surface of a vesicle

allows the vesicle to dock on its particular target membrane

Rab and tethering proteins

provide the initial recognition btwn a vesicle and its target membrane

a v-SNARE on the vesicle then binds to a complementary t-RNARE (complementary SNARE proteins)

ensure that transport vesicles dock at their appropriate target membranes and catalyze the final fusion of the two membranes

SNARE proteins catalyze the fusion of the vesicle and target membranes

• the tight pairing of v-SNAREs and t-SNAREs draws the two lipid bilayers into close apposition, squeezing out any water molecules that remain trapped btwn the membranes and allowing their lipids to flow together to form a continuous bilayer

• after fusion, the two SNAREs are pried apart to be used again

glycosylation of proteins on asparagines (in the ER)

• when an asparagine in a growing polypeptide enters the ER, it is glycosylated by the addition of a branched oligosaccharide chain

• each oligosaccharide chain is transferred as an intact unti to the asparagine from a lipid called dolichol, catalyzed by oligosaccharyl transferase

• asparagines that are glycosylated are always present in the tripeptide sequences asparagine-X-serine or asparagine-X-threonine

accumulation of misfolded proteins in the ER lumen triggers…

unfolded protein response (UPR)

transmembrane sensor proteins

recognize misfolded proteins and activates a different component of the UPR

can either stimulate the production of transcription regulators, or inhibit protein synthesis

tagging a protein with GFP function

allows the resulting fusion protein to be tracked throughout the cell

tagging a protein with GFP

at high temps, the GFP fusion protein labels the ER

as the temp is lowered, the GFP fusion protein rapidly accumulates at ER exit sites and then moves to the golgi apparatus

the fusion protein is finally delivered to the plasma membrane

constitutive secretory pathway

• in all eukaryotic cells

• secretes soluble proteins

• supplies the plasma membrane with newly made lipids and proteins

regulated exocytosis pathway

• in specialized secretory cells

• selected proteins in the trans golgi network are diverted into secretory vesicles, where proteins are concentrated and stored until an extracellular signal stimulates their secretion

pinocytosis

takes in fluids and solutes

phagocytosis

takes in larger substances like bacteria

LDL enters cells via receptor mediated endocytosis

• LDL binds to LDL receptors on the cell surface and is internalized in clathrin coated vesicles

• the vesicles lose their coat and fuse with endosomes

• in the endosome’s acidic environment, LDL dissociates from its receptors and ends up in lysosomes where it is degraded to release free cholesterol

• the LDL receptors are returned to the plasma membrane via transport vesicles to be used again

viruses entering cells via receptor mediated endocytosis

vesicles will fuse with lysosomes where the low pH will allow the release of the viral genome into the cytoplasm

three pathways from the endosomal compartment in an epithelial cell:

• recycling

• degradation

• transcytosis

three pathways (expanded) in an epithelial cell

• tight junctions separate the apical and basolateral plasma membranes, preventing their resident receptor proteins from diffusing from one domain to another

• retrieved receptors are returned either to the same plasma membrane domain from which they came (recycling) or to a different domain (transcytosis)

• receptors that are not specifically retrieved from early endosomes follow the pathway from the endosomal compartment to lysosomes, where they are degraded

• if the ligand that is endocytosed with its receptor stays bound to the receptor in the acidic environment of the endosome, it will follow the same pathway as the receptor or will be delivered to the lysosomes for degradation

the lumen of the lysosome is maintained at an acidic pH by…

an atp driven H+ pump that hydrolyzes ATP to pump h+ into the lumen