intracellular transport

Adv Cell Bio

protein sorting

synthesized by ribosomes in cytosol but are directed by a sorting signal

protein translocation (transport thru membrane between topologically distinct regions)

gated transport (NPC)

vesicular transport (membrane-enclosed between topolically equivalent regions)

engulfment (membrane sheets wrap around cytoplasmic regions)

intracellular digestion - autophagy

autophagosomes form via engulfment

fuse with lysosome for degradation

recycle macromolecules

• regular cellular maintence

• prominent in development and differentiation

intracellular digestion - lysosomes

filled with acid hydrolases (function at low pH)

• vacuolar H ATPase maintains low pH

• vacuoles are specialized lysosomes in plant, fungi

pathways to the lysosome

autophagy

endocytosis - can ingest membrane components, extracellular solutes/molecules, whole cells

• pinocystosis (constant in most cells)

• phagocytosis (specilized cells)

can also be sorted to Golgi or recycled back to plasma membrane

protein sorting - vesicular transport

topologically equivalent

cargo does not cross membrane

maintain orientation to cytosol

selective budding and fusion

• protein and lipid markers, protein machinery

intracellular sorting

establishes cellular compartment identity

can respond to the environment

add/remove cell-surface proteins

• receptors

• ion channels

• transporters

change lipid composition of plasma/organelle membranes

endocytic pathway

moves from extracellular space to plasma membrane into the cell/cytosol 

secretory pathway

moves from ER, golgi, to plasma membrane

Golgi apparatus/complex function

main site of carbohydrate synthesis

polysaccharides

• plant cell wall: pectin, hemicellulos

• animal extracellular matrix: glycosaminoglycans

oligosaccharide chains

• glycoproteins

• glycolipids

Golgi structure

series of flattened membrane enclosed compartments (cisternae)

• 4 to 6 per Golgi stack in animals

Cis face = entry face, coming from Er

trans face = exit face, bound for plasma membrane or endosome

each compartment contains unique glycosidases and glycosyl transferases 

• all single pass transmembrane proteins (not soluble like ER lumen proteins)

glycoproteins are successively modified

vary by cell type and developmental stage

movement thru Golgi

cytoplasmic Golgi matrix proteins organize Golgi architecture

• scaffold adjacent cisternae

• provide structural integrity

Golgins extend from each compartment to recruit the correct vesicles

vesicle coat proteins

coated vesicles bud off from specialized, coated regions of membranes (distinctive cage of proteins on cytosolic surface)

clathrin coated - originated from Golgi, endosomes, plasma membrane

COPII coated - originate from ER

COPI coated - originate from Golgi, retrograde back to ER

retromer coated - originate from endosomes

clathrin coated

clathrin protein = 3-legged structure

outer layer of coat

assembles hexamers and pentamers to form a cage around vesicle

introduces membrane curvature

coat assembly - clathrin 

adaptor proteins form inner layer of coat

• bridge clathrin and transmembrane proteins

cargo receptors bind cargo molecules, adaptor protein recruits clathrin , membrane ebnding and fission proteins form vesicle and pinch off

membrane identitiy: phosphoinosidtides

PI < 10% of membrane phospholipids but important for signaling

can be phospohrylated at 3’, 4’ or 5’ position of inosital sugar = PIP

Pi and PIP kinases add phosphates

PIP phosphatase remove phosphate

PIP binding proteins recognize specific PIPs

different compartments contain different Pi/PIP kinsases and phosphastases

• PIPs are highly regulated

• specifically mark different organelles and even portions of membrane of a single organelle

coat assembly with PIP

AP2 adaptor protein specifically binds Pi(4,5)P2

conformational change exposes binding site for cargo receptors in membrane

binds clathrin and induces membrane curvature

coat assembly - COPII

triggered by GEF in ER to recruit adaptor proteins

activates GTPase to form bud

concludes when GTP is hydrolyzed

pinching off vesicles

actin polymerization (uses ATP hydrolysis) drives vescicles away from original membrane

dynamin uses GtP hydrolysis to pinch off vesicle

uncoating

Clathrin coat disassembles after budding (PIP phophatase → adaptor protein release)

COPII coat disassemble at target membrane (kinase at destination phosphoryaltioncoats)

COPI uncoating triggered by curvature of vesicle (GAP senses curvature)

membrane ID: Rab proteins

guide transport vesicles

Rab GTPases:

• GDP bound = inactive, soluble in cytosol

• GAP triggers GTP hydrolysis

• GTP bound = active, membrane bound

• anchored by lipids modiciation

• membrane bound GEF activates

membrane bound Rab binds Rab effector proteins

• tethering proteins on target membrane

• motor proteins to transport vesicle

• SNARE proteins to mediate membrane fusion

membrane fusion

must bring lipid bilayers into close proximity 

• lipids flow between two membranes in close proximity

• need to displace H2O from hydrophilic phospholipid head (energetically unfavorable!)

• hydration shell prevents random membrane from fusing

SNARE proteins = fusion proteins that provide force to bring bilayers together and displace H2O

• V-SNARE on vesicle membrane. single polypeptide chain

• T-SNARE on target membrane, usually 3 protein complex

• highhly specific paring

• helical zipper, very stable 4 chain bundle = highly energetically favorable = energy displaces H2O

after fusion, SNARE complex is pried apart and recyeld

• requires AtP hydrolysis