Unit 5

Phospholipid Structure

consists of polar head group and two nonpolar fatty acids; diaglycerols with polar head groups attached via a phosphate

Inositol Ring

is phosphorylated in signaling pathways; 3,4, and/ or 5 positions

Phosphatidylinositols (PIPs)

act as binding sites for tethers in vesicle fusion

Triacylglycerols

extremely hydrophobic and form lipid droplets surrounded by phospholipid monolayer shells

Protein Sorting

happens in the cytosol

Smooth ER

diacylglycerol phospholipids are synthesized on the cytosolic side of the membrane from cytosolic precursors

Secretory vesicles

a membrane-enclosed sac that transports proteins from the golgi to the cell surface

Palade Pulse chase experiment

purpose is to trace how newly made proteins move through a cell; cells were pulsed with radioactive amino acids and then chased with normal ones

What happened in the pulse-chase experiment?

Immediately following the pulse, labeled proteins were in the rough ER, then after 7 minutes they were in the golgi apparatus, and later they were found in secretory vesicles and eventually outside the cell

Secretory Pathway

Rough ER, golgi, secretory vesicles, plasma membrane, outside of cell

Topology of the secretory system

lumen of the ER is topologically equivalent to the outside of the cell; whatever side faces the ER lumen will face the extracellular space following secretion; the orientation of membrane proteins is set in the ER and stays the same as vesicles move through the pathway; cytosolic domains remain in cytosol; lumenal domains stay inside vesicles; lumenal domains are expressed on the outside surface of the PM when vesicles fuse

Steps of Co-translational translocation of proteins into the ER

signal peptide appears from ribosome, SRP binds the signal and temporarily stops translation, SRP receptor on ER membrane binds SRP and brings ribosome to translocon; SRP and its receptor use GTP energy to release their ribosome and translation resumes; the growing protein is threaded into the ER through the translocon channel; signal peptidase cuts off the signal peptide, and the protein folds in the ER

Signal peptide

found at the amino N-terminus; 15-20 amino acids long (a helix); has 1-2 positive basic residues followed by a hydrophobic core and directs proteins to the ER

Signal Recognition Protein/particle

RNA-protein complex that binds the signal peptide and pauses translation until the ribosome binds to the ER

SRP receptor

ER membrane protein that recognizes SRP and guides the ribosome to the translocon

Translocon

Sec61 complex; protein channel in the ER membrane where the new chain enters the ER lumen or inserts into the membrane; has a plug to close the pore when not in use

Signal peptidase

enzyme inside ER that removes the signal sequence from the new protein

BiP

a chaperone that helps fold proteins correctly inside the ER; keeps unfolded proteins from leaving

Flippases

move new phospholipids from the cytosolic side of the ER membrane to the lumen side to balance membrane growth

Protein transmembrane domains

19-21 hydrophobic amino acids flanked by basic residues; + charge

Stop transfer sequence

dissociates ribosome from translocon

Type 1 protein

exactly like secreted proteins, except the co-translational translocation stops after the insertion of the TMD (ex- LDL receptor); carboxy terminus in cytosol, amine terminus in lumen

Type 2 protein

translation starts in cytosol, then stops after expression of signal sequence, then co-translational translocation proceeds; amine terminus in cytosol and carboxy terminus in lumen; some proteins have internal signal sequence not cleaved (ex- transferrin receptor)

Multi-span proteins

alternate internal signal sequences and stop transfer sequences

N-linked oligosaccharides

sugars are added to asparagine in the sequence while the protein is entering the ER; the initial chain has 14 sugars and is trimmed and modified in the ER and golgi; these sugar chains help proteins properly fold, preventing clumping and later acting as sorting signals

Unfolded Protein Response

large amounts of unfolded proteins trigger a signal cascade that inhibits translation and activates transcription

ERAD

Endoplasmic Reticulum-Associated Degradation; misfolded proteins are recognized and destroyed by ubiquitin-proteosome system

Steps of ERAD

calnexin and calreticulin help glycoproteins fold; if folding fails UGGT re-adds a glucose so the protein can try again; if it stays misfolded for too long mannose residues are trimmed; proteins with trimmed mannose are bound by OS9/XTP2B and pulled back to the cytosol; in the cytosol, they are ubiquitinated and degraded by the proteasome

GPI anchors

glycosylphosphatidylinositol anchor; added in ER by GPI transamidase, replacing the protein’s original C-terminal membrane segment; these proteins face the ER lumen and later the outside of the cell, and the GPI anchor helps target some to specific membrane regions

KDEL

soluble ER proteins have a KDEL sequence at the end (Lys-Asp-Glu-Leu); keep things in ER and retrieve things from Golgi; have a higher affinity at a lower pH

What happens if ER proteins accidentally leave the ER?

KDEL receptors in the golgi or ERGIC recognize them and return them to the ER via retrieval vesicles

Movement of proteins through golgi

cis to medial to trans then sorted in vesicle carriers

Cis-Golgi

entry side facing the ER; early sugar and lipid modification begin

Medial-Golgi

middle area; further modification of sugars and proteins

Trans-Golgi

final processing and sorting center where proteins are packed for lysosomes, secretion, or the plasma membrane

COPll

moves from ER to golgi (forward/ anteriograde)

COPl

moves from golgi to golgi or ER (backward/ retrieval)

Clathrin

moves between trans-golgi, endosomes, lysosomes, and plasma membrane

Coats

form vesicles and select cargo

8 steps of membrane trafficking

initiation, budding, scission, docking, tethering, uncoating, fusion, and recycling

1) Initiation

small GTPases (Arf proteins) become active and recruit adaptor proteins; adaptors bind to cargo and coat proteins

2) Budding

adaptor proteins collect cargo and coats self-polymerize and form the vesicles

3) Scission

pinchases/dynamin uses GTP to pinch the vesicle off the membrane

4) Docking

Rabs- small GTP binding proteins, different ones on different vesicles

5) Tethering

SNAREs; the Rab proteins on the vesicle interact with tethering factors on the target membrane

6) Uncoating

the vesicle coat is removed once it reaches the target membrane; GTPase, ARF hydrolyzes GTP and coat falls apart

7) Fusion

SNARE-pins; SNARE protein on the vesicle (v-SNARE) and target (t-SNARE) zip together, fusing the membranes

8) Recycling of the SNAREs

uses NSF, SNAPs, and ATP to separate SNARES; NSF/a-SNAP

SNARE activation

proteins (Munc18) keeps the t-SNARES and v-SNARES apart and inactive until tethers bind to the correct docking molecules, then the SNAREs are brought together, activated, and SNARE-pins drive fusion

Lysosomes function

the cell’s recycling center; breaks down proteins, lipids, carbs, and nucleic acids; can get cargo to degrade from phagocytosis and autophagy

Autophagy

the degradation of cytoplasmic proteins and organelles by their enclosure in the cytoplasmic vesicles that fuse with lysosomes

Autophagasome

a vesicle containing internal organelles enclosed by fragments of cytoplasmic membranes that fuses with lysosomes

Autolysosome

a structure formed by the fusion of a lysosome and an autophagosome containing damaged and/or intracellular substances during the process of autophagy

Lysosome pH

contain acid hydrolases that only work at acidic pH (5); the acidic environment is maintained by an ATP-driven proton pump

What are the two roles for pH in a lysosome?

activates lysosomal enzymes and keeps the cytosol safe (enzymes won’t work if they leak out (cytosol pH = 7.2))

Mannose-6-phosphate (M6P)

added to lysosomal enzymes in the cis-Golgi recognized by M6P receptors in the trans-Golgi, which packages them into vesicles that deliver them to lysosomes; high affinity for lysosomal enzymes at pH 6, low affinity at pH 5

Apical Domain

exposed free surface of polarized epithelial cells; absorption of nutrient

Basolateral Domain

the surface region of a polarized epithelial cell that is in contact with adjacent cells or the ECM; transfer of nutrients into blood

Post-translational modifications

signal peptide cleavage, disulfide bond formation, n-linked glycosylation, o-linked glycosylation, mannose-6-phosphate tagging

Signal Peptide Cleavage

occurs in the ER; the signal peptide is cut off by signal peptidase, an enzyme on the lumenal side of the ER membrane; removing it allows the protein to fold into its proper shape and prevents it from being stuck on the membrane\

Disulfide Bond Formation

occurs in the ER; forms covalent bonds between cysteine residues; helps stabilize the 3D structure of secreted and membrane proteins

N-Linked Glycosylation

occurs in ER and moves to Golgi; in the ER, a 14-sugar oligosaccharide is attached to an asparagine residue and partially trimmed while chaperones assist in protein folding; in the golgi, this gylcan is further modified to guide protein transport, enhance stability, and prevent degradation; 1) complex oligosaccharide synthesized on dolichol 2) complex oligosaccharides transferred to Asn 3) complex oligosaccharides trimmed- glucose removed

O-Linked Glycosylation

occurs in Golgi; in the Golgi, individual sugars are added one by one to serine or threonine residues, forming O-linked glycosylation common in secreted proteins and proteoglycans; this modification increases solubility, provides cell surface protection, and aids in cell recognition

Mannose-6-Phosphate Tagging

occurs in cis-golgi; in cis-golgi, lysosomal enzymes are tagged with M6P, which is recognized by M6P receptors in the trans-Golgi to direct them into vesicles bound for lysosomes; this modification ensures proper enzyme delivery, and defects in the process cause enzymes to be secreted outside the cell instead of reaching lysosomes