BICD 110 midterm 1

LECA

hypothetical last common ancestor of all eukaryotes (around 1.5-2 billion years ago)

biological scales

mammalian cells: 10-100um

prokaryotic cells: 1-10um

organelles: 2-20um

micromolecular complexes: 200nm-2um

proteins/ ribosomes: 20nm

Hooke

invented compound microscope

discovered plant cells, named “cells” after cork micrograph drawing

Leeuwenhoek

invented single lens microscope

discovered microorganisms “animacules”S

Schleiden

“every part of a plant is made of cells”

Schwann

“plants, animals, and their products are made of cells”

Virchow

created cell theory

Palade

used diamond knife + electron microscopy to visualize many structures

invented pulse-change, SDS-PAGE

cell theory

1. all living organisms are 1+ cells

2. the cell is the most basic unit of life

3. all cells arise from pre-existing cells

visible light spectrum

450nm: blue

500nm: green

600nm: orange

650nm: red

fluorophores

fluorescent chemicals that can re-emit light by dropping down from an excited state to ground state

SEM (scanning electron microscopy)

samples are processed w/ metals and electrons scan surface

allows surface features to be visualized/ topographical map to be made

TEM (transmission electron microscopy)

samples are heavily processed w/ metals (thick and thin samples can be prepared); electrons pass everywhere but metal areas to visualize structures w/in cells

cryo-EM

visualizes samples more naturally; samples frozen rapidly to form non-crystalline ice

immunolabeling

antibodies respond to a protein of interest and have covalently attached labels (fluorescent dyes or gold particles)

primary antibodies + secondary antibodies to amplify signal

fixation

required for electron microscopy/ immunolabeling

freezes cell structures in place/ prevents damage during processing for visualization

permeabilization

makes plasma membrane permeable to reagents for labeling/ staining for visualizaion

four concepts for the chemistry of life

1. molecular complementarity: complementary interactions (shape, polarities, etc.)

2. polymerization: combination of subunits to create emergent properties

3. chemical equilibrium: rxn equilibriums can shift based on diff. environments

4. energy: most common source of energy is ATP

bond strengths/ energy listed in order

thermal energy < van der waals < h-bonds < ATP bond < C-C < C=C

covalent bonds x10-100 strength over noncovalent

hydrophobic effect

hydrophobic molecules group together in water to decrease entropy

T_m of lipids

melting temp of lipids; the temp at which 50% of lipids in a membrane are fluid

types of phospholipids

glycerophospholipids and sphingolipids

glycerophospholipids

• phosphoatidylcholine (PC)

• phosphatidylserine (PS)

• phosphatidylethanolamine (PE)

• phosphatidylinositol (PI)

• phosphatidylglycerol (PG)

• cardiolipin (CL)

sphingolipids

• sphingomyelin (SM)

cholesterol

acts as fluidity buffer in lipid membranesho

homeoviscous adaptation

ability of cells to change their membrane lipid composition to control membrane fluidity

(introduction/ elimination of kinks/ unsaturated bonds, shorter/ longer acyl chains)

protein hierarchical structures

1. primary (linear sequence of AAs)

2. secondary (alpha helixes, beta sheets, held together w/ backbone h-bonds)

3. tertiary (3D protein shape)

4. quaternary (multiple tertiary structure peptides)

5. supramolecular complexes (multiple subunits associating w/ one another; 10s-100s)

Plant cell unique structures

Cell wall to maintain cell’s shape

Vacuole for water storage

Chloroplasts for photosynthesis

Plasmodesmata as cell junctions

Animal cell unique structures

Microvilli to increase SA for absorption of nutrients from surroundings

Single bilayer organelles

ER, ERGIC, golgi, TGN, PM, lysosomes, peroxisomes

Two bilayer organelles

Mitochondria/ chloroplasts, nucleusM

Monolayer organelles

Lipid droplets

How the first membranous organelles formed

Infolding of PM → nucleus + ER

Engulfing of another prokaryote → mitochondria + plastid (chloroplasts)

ER % membrane surface

Smooth: 10%, Rough: 50%

Combined 50-60%

ER % volume of cell

Smooth: 6%, Rough: 10%

(inner) Mitochondrial membrane % membrane surface

20-30%

Mitochondria % cell volume

20%

Cytosol % cell volume

50%

Rough ER morphology + function

sheet structure, enriched near nucleus/ golgi

secretory pathway for proteins, site of protein folding/ post-translational modifications, protein quality control, N-glycosylation

Smooth ER morphology + function

tubular structure evenly distributed through cytoplasm

steroid hormone synthesis, cellular detox center, calcium ion storage

ERGIC morphology + function

space between ER/ golgi; contains vesicles

sorts cargo for reconsumption by ER or consumption to golgi

Golgi morphology + function

stacked sacs (cisternae) with a cis face toward rough ER and trans face toward PM

flat centers w/ concentrated enzymes where reactions take place and fenestrated regions where transport takes place (COPI and COPII vesicles)

O-glycosylation, remodeling of N-glycans, stepwise modification of cargo, lipid synthesis/ transport, lipidation of proteins

TGN morphology + function

tubular system at the trans end of golgi apparatus

recieves cargo from endosomal system/ golgi, trasport of lysosomal enzymes to lysosomes/ endosomes, processing of proproteins, retrograde transport of golgi residents, calcium-dependent sorting (regulated/ constitutive secretion)

PM morphology + function

lipid bilayer with proteins throughout (fluid mosaic model)

physical barrier w/ selective permeabiilty, transport of solutes, endocytosis/ phagocytosis/ exocytosis, cell signaling

endo-lysosomal system morphology + function

network of interconnected vesicles/ tubules

endocytosis, phagocytosis, autophagy for digestion/ recycling using lysosomes

nucleus morphology + function

contains nuclear outer/ inner membranes, nucleolus, nuclear pores, continuous w/ ER

stores DNA, separates transcription from translation, selectively permeable

mitochondria morphology + function

outer/inner membrane with crystae for SA, filled with matrix/ matrix granules

ATP production, oxidation of fatty acids, citric acid cycle, DNA storage

lipid droplets morphology + function

organelles w/ a core of neutral lipids enclosed in phospholipid monolayer

energy storage to maintain homeostasis/ membrane homeostasis

peroxisomes morphology + function

lipid bilayer with crystalline core

generation/ scavenging of reactive oxygen species, breakdown of “difficult” molecules, biosynthesis of special membrane lipids

SDS-PAGE

technique by palade; uses gel as a sieve to separate proteins based on size

proteins are denatured + coated w/ ionic (negatively charged) detergent

pulse-change experiments

by palade; provides cells w/ a short pulse of radiolabeled AAs to observe where the newly synthesized proteins go over time

Blobel/ signal sequence hypothesis

proteins contain signal sequences that can direct them to the rough ER

Process: co-translational insertion of secretory proteins into the ER

1. ER signal sequence emerges from ribosome during protein synthesis

2. signal recognition particle binds the signal sequence/ pauses protein synthesis

3. the SS-SRP-ribosome complex binds to the SRP receptor which connects the complex to the translocon (sec61) using GTP

4. SRP dissociates and an additional GTP is used to line the protein up with the translocon channel

5. ribosome unpauses protein synthesis and continues building through translocon into ER lumen

6. SS sequence is cleaved by signal peptidase and is rapidly degraded

7. translation completes and protein is drawn the rest of the way into the lumen/ folded as the translocon closes

sec61

translocon

hourglass-shaped channel shaped by isoleucine (mimics hydrophobic membrane to interact with hydrophobic signal sequence)

lateral gate for transmembrane proteins to exit into the membrane

“plugged” gate

Process: membrane protein insertion to ER membrane

1. protein is connected into the translocon

2. hydrophobic stop-transfer anchor sequence enters the translocon channel during transcription

3. anchor sequence is transferred through lateral gate into membrane and translocon closes (positively charged is always in cytosol)

4. synthesis of the protein continues until the stop codon is reached

type I membrane protein

signal sequence at N-terminus, N-terminus in ER lumen and C-terminus in cytosol

type II membrane protein

C-terminus in ER lumen and N-terminus in cytosol

type III

N-terminus in ER lumen and C-terminus in cytosol

type IV

multiple anchoring regions in the membrane; weaving/ in out with N and C terminus on opposite sides of the membrane

tail-anchored proteins

C-terminus is embedded in membrane, N-terminus in cytosol

GPI-anchored proteins

tail anchored proteins are built with N-terminus in ER, then transferred off anchor region onto a GPI-molecule anchor

Process: N-glycosylation

occurs during protein synthesis through the translocon

• N-glycan precursor (oligosaccharide) is made of 2 n-acetylglucosamine (GlcNAc), 9 mannose residues (Man), and 3 additional sugars (Glc) → (Glc)₃(Man)₉(GlcNAc)₂

• Oligosaccharides are added to asparagines in (Asn - x - Ser/Thr) regions on target proteins as they emerge through the translocon

• Addition/ removal cycle of glucose residue plays a role  in protein folding

• Removal of one mannose residue

• final product for cell export: (Man)₈(GlcNAc)₂

CNX/CRT

“chaperones” of correct protein folding in the ER that bind w/ the addition of glucose onto n-glycan regions and retain proteins in ER until properly folded

OS-9

if protein is continuing to be misfolded, mannose are trimmed and OS-9 exports misfolded protein for disposal

PDI

contains S-S disulfide bond that locates two “S” in the protein and transfers the disulfide bond to between them

as the protein continues to emerge, PDI transfers/ breaks bonds until the most thermodynamically stable

BiP

chaperone protein that binds to exposed regions of growing protein chain and stabilizes them until a protein is folded correctly

in influenza, activates Ire1 transmembrane proteins to dimerize and trigger other chaperones

T_s mutant genes

genes which at a permissive temp (lower), the protein forms normally, but at a restrictive temp (higher), the protein doesn’t fold

Necessary things for vesicular transport

1. machinery to attract a coating

2. coat proteins (w/ rigid form, deforms membrane to match, stabilizes the carrier)

3. uncoating of carrier (mechanism that takes coating away to allow for fusion)

4. machinery for fusion (specificity for the target membrane, ability to pull carrier/ target membrane together)

COPII vesicles

anteretrograde - from ER to golgi/ ERGICC

GAP, GTPase, GEF reationship

GAP makes GTPase more effective/ expidited

GEF exchanges GDP for GTP on GTPase to change from inactivated to activated

COPII vesicle biogenesis

1. Sar1-GDP (inactivated GTPase+GDP complex) interacts with Sec12 (GEF activity) on the ER membrane to activate Sar1

2. Sar1 integrates itself into the membrane on the cytosolic-facing side

3. Sar1-GTP recruits sec23/ sec 24 coat protein complex and sec 13/ sec 31 completes the coat assembly (double coating)

4. Coat curves and pinches off ER membrane region

5. Sec23 GAP activity stimulates Sar1-GTP hydrolysis, re-inactivating Sar1 and causing it to detach from the membrane along with the coating

** additional factors needed for bulkier cargo (animal cells)

COPI vesicles

retrograde - from golgi/ ERGIC to ER

COPI vesicles biogenesis

1. p24 attracts Arf1-GDP (inactivated GTPase + GDP complex) which interacts with GBF1 (GEF) in the cytosol to activate Arf1

2. Arf1-GTP recruits a coatomer that assembles on membrane and pinches off the vesicle

3. ArfGAP activity stimulates Arf1-GTP hydrolysis

4. Release of Arf-GDP from vesicle membrane causes disassembly of the coating

COPII/ COPI fusion mechanism

1. V-snares located on vesicles form coils with t-snares on target membrane 1 v-snare + 3 t-snares)

2. coiling of snares brings membranes close together for docking/ fusion

3. NSF ATPase separates the helix bundle after fusion occurs

COPII vesicle - cargo signals for proteins

transmembrane: Inner layer (sec23/ sec24) of COPII coat recognizes di-acidic + di-hydrophobic signals on cargo transmembrane proteins

other: ERGIC-53 binds high mannose n-glycans (Man₈(GlcNAc)₂) made in the ER to cluster them into COPII carriers; binding is pH-dependent and releases in acidic ERGIC environment

COPI vesicle - cargo signals for proteins

transmembrane: Coatomer of COPI recognizes basic signals on cargo transmembrane proteins

other: KDEL receptors bind to missorded ER-resident proteins in the ERGIC to incorporate them into COPI vesicle

Maturation model of intra-golgi transport

COPI vesicles move enzymes to former cisternae as they move forward and convert themselves from cis to trans; cargo stays stationary

Evidence: cis and trans golgi converted colors (cis protein color → trans protein color) over time in yeast cells

Stable cisternae/ vesicular transport model of intra-golgi trasport

Cisternae are stable compartments w/ a specific set of resident enzymes that stay put; cargo is transported between them w/ tubular or vesicular carriers

Evidence: In human cells over time, cis/ trans parts of the golgi were always cis/ trans

Process: Processing of N-glycans in Golgi

• Cis golgi: mannosidases remove 3 man residues

• Medial golgi: 1GlcNAc added, 2 more mannoses removed, 2 more GlcNAc added, 1 fucose added

• Trans golgi: 3 gal (galactose) residues added

** some proteins selectively choose some steps and not other steps

O-glycosylation

occurs in the cis-golgi

O-glycan precursor binds to Ser/ Thr residues

Temperature-sensitive VSV G experiment

Golgi system was set up in vitro and injected w/ a temperature sensitive, radioactively labeled virus G protein

Misfolded protein under restrictive temps was retained in ER and properly folded protein under permissive temps moved through secretory pathway + could be observed

lipid composition throughout the cell

• closer to PM: more rigid, more cholesterol/ sphingolipids

• closer to interior: more fluid/ flexible, more phosphoacytylcholine

OSBP

feeds cholesterol from the ER where it’s produced into the trans-golgi PM

clathrin-coated vesicles (structure)

bud from TGN and PM for transport

3 heavy + 3 light chains = triskelions

36 triskelions = complete clathrin coating

5 types of cargo/ destinations for TGN to sort

1. retrograde transport of golgi enzymes to trans-golgi (GOLPH3)

2. lysosomal enzymes into lysosomes

3. lysosomal enzymes into late endosomes

4. constitutive secretory vesicles

5. regulated secretory vesicles

process: CCV biosynthesis

• Arf1-GDP binds to cytosolic GEF and is activated

• Arf1-GTP binds to TGN membrane, recruits cytosolic AP-1 (adapter protein) to the membrane

  • Arf1-GTP induces conformational change in AP-1 to open and attract cargo sorting motifs

• Clathrin (coat proteins) are recruited by AP-1 and bind to form coating

• Dynamin (GTPase) polymerizes and uses GTP to pinch off the membrane

• Hsc70/ auxilin use more GTP to break down the clathrin coating on vesicle exterior and allow vesicles to be ready for fusion

TGN cargo signals for proteins (general proteins, golgi residents, lysosomal proteins)

general proteins:

• u1 subunits: binds tyrosine+hydrophobic (YXX(hydrophobic)) regions on proteins

• o1-y subunits: binds dileucine (LL) regions on proteins

golgi residents:

• GOLPH3: recognizes golgi-resident protiens and confines them to COPI vesicles for retrograde transport back to the golgi

lysosomal proteins:

• M6P receptor: binds to m6p on modified lysosomal enzymes and attracts clathrin coating

Process: TGN sorting lysosomal enzymes

Pre-TGN, in the cis-golgi:

• GlcNAc phosphotransferase recognizes QS/HEY sequences in newly synthesized lysosomal enzymes + transfers phosphorylated GlcNAc to 1+ mannose residues

• Phosphodiesterase removes GlcNAc group to leave a mannose 6 phosphate (M6P) on the lysosomal enzyme

TGN:

• M6P receptor is active in the higher pH at the TGN membrane and recruits APs to create a clathrin coating

• Clathrin coating is broken down w/ hsc70/ auxilin and can fuse with late endosome/ lysosomes (lower pH = M6P receptor releases enzyme)

• M6P and vesicle can be recycled back to TGN and PM

Regulated exocytosis

requires a signal for exocytosis to occur; calcium dependent sorting

seen in synaptic vesicles and release of insulin by pancreatic beta cells

Constitutive secretory pathway

constantly secreting; calcium dependent sorting

TGN processing of proproteins

constitutive pathway:

• Furin endoprotease cleaves peptide at c-terminal end of two consecutive AAs

regulated secretory pathway:

• PC2 and PC3 endoproteases cleave central region of insulin

• Carboxypeptidase cleaves two c-terminal basic AA residues