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209 terms

ANAT262 - Post Midterm Stuff

McGill Winter 2012
STUDY
PLAY
cell-free system
used to study particular aspects of a process without all of the complexities of a fully intact cell. To the extent possible, the process will be reduced to its minimal components
cell-free system
bottom up way of looking at a cell; look at a process without anything that could possibly interfere
NSF
separtes SNAREs thereby allowing for transport to occur
NEM
reacted with proteins to inactivate the transport assay
NEM
treatment with this molecule resulted in accumulation of vesicles which were mostly not coated
VSV-G
cargo protein used in the in vitro system for intra-Golgi trafficking
NEM
inhibits transport
NSF
inactivated NEM and rescued transport
NSF
required for SNARE-mediated membrane fusion
GTP-gamma-S
blocks GTP hydrolysis by trimeric G proteins, Ras family GTPases, and other GTPases. Also blocked the transport assay. (analog)
GTP
regulates an uncoating step
NSF
regulates a fusion step
Arf1
has to hydrolyze its GTP to release the coat.
NSF
interacts with SNAREs to regulate fusion of the vesicle with Golgi membrane
SNAREs
perform the actual fusion event
NSF
NOT required for vesicle formation; required only for fusion
GTP-gamma-S
non-hydrolyzable analog of GTP
Arf1
GTPase required for the uncoating of COPI vesicles
microsomes
fragmented rough ER membranes used in the Rothman lab
Sar1
GTPase required for the uncoating of COPII vesicles
DGAT
synthesizes triglycerides
Lipid Droplet
form in any cell type; belong to the cell; facilitate in ER to cytoplasm transport of fatty acids and triglycerides
Lipoprotein Particle
form in specialized cell types only; secreted from these special cell types; enterocytes and hepatocytes
ACAT
synthesizes cholesterol enzymes; found in the ER membrane; faciliates exit of cholesterol from ER
ACAT
removes the polarity of cholesterol by adding a hydroxyl to its structure
DGAT
catalysis of the last step in the production of trigylcerides
Triglycerides
highly insoluble, like cholesterol esters, and must by sequestered into lipoprotein particles or lipid droplets
Lipid Droplets
released in a phospholipid monolayer into the cytoplasm
Lipoprotein
releases in a phospholipid monolayer into the ER lumen
LDL
bad cholesterol
HDL
good cholesterol
Chylomicrons
secreted by enterocytes and cells of the small intestine; transfer lipid into bloodstream; get cholesterol into a bloodstream;
HDL
secreted by the liver; almost no lipid; picks up cholesterol and carries it back to the liver
LDL
secreted by the liver; produced by stripping triglycerides from VLDL particles
ApoA
found in HDL
ApoB48
found in chylomicrons; lacks region for binding the LDL receptor
ApoB100
can bind to LDL receptor
ApoC
small lipoprotein found in HDL and other lipoprotein molecules (sometimes)
ApoE
found in chylomicrons; binds to receptor in the liver
Apolipoproteins
wrap around the lipoprotein
ApoB
really big lipoproteins; can form lipoprotein with only one of these
Chylomicrons
secreted by enterocytes of the small intestine; contain ApoB48, which cannot bind to LDL receptor
Liver
can accumulate cholesterol and triglycerides from several sources
cholesterol
can be re-secreted in VLDL particles, stored in lipid droplets in hepatocytes, or converted to bile salts; cannot be destroyed
VLDL
cholesterol and triglycerides in liver (delivered by chylomicrons or HDL) can be secreted as this
VLDL
contains ApoB100, which can bind LDL receptor
VLDL
will shrink as lipoprotein lipase removes triglycerides
VLDL
gets stripped of triglycerides by lipoprotein lipase and gets smaller
LDL
can be taken up into cells by binding LDL receptor
degraded LDL
can be accumulated by binding to "scavenger receptors" on macrophages
macrophages
can be overloaded and tranformed into pathogenic "foam cells" or even killed in artery walls
HDL
ApoA is the major lipoprotein; endocytosed by the liver; acts as a sponge for triglycerides; transfer cholesterol from cells to liver
Lipid Droplets
major role is storage of lipids; core of cholesterol esters (ACAT) and triglycerides (DGAT);
PAT
proteins playing a structural role in stabilizing the surface of the lipid droplet; can be phosphorylated by Protein Kinase A (PKA)
Hormone Sensitive Lipase
can bind to phosphorylated PAT proteins, where it can release free fatty acids from triglycerides; emergentcy mechanism
ATGL
another lipolytic enzyme that can bind to PAT proteins on lipid droplets. its regulation is unclear
cAMP
activation of the beta-adrenergic receptor on cells activates this; acts on PKA
PKA
activates HSL or PAT Proteins
Lipophagy
variant of autophagy making possible the transfer of lipid droplets to lysozomes
Endocytic Vesicle
delivers cargo from cell surface into cell
Vesicles
fuse with early endosomes and deliver their cargo; clathin and non-clathrin coated
Early Endosome
acidic due to a proton pump (Vacuolar H+ ATPase) in membrane; Many ligands detach from receptors here do to low pH; function is to sort
Early Endosome
FIRST major component of endocytic pathway; also known as "sorting endosomes"
Recycling Endosome
tubular compartment that carries receptors and membranes back to the cell surface;
Multi-Vesicular Body
aka "late endosome;" matures from early endosome; contains internal vesicles visible by electron microscopy
Internal Vesicles
membrane proteins targeted for degradation are budded into these
Multi-Vesicular Body
degradation does not take place here efficiently
Lysosome
final degradative compartment. Acidic. Dense appearance by electron microscopy. contains an abundant amount of degradative enzymes and channels to transport amino acids and other degradative products into the cytoplasm
degradative enzymes
work only at acid pH; if the lysosome were to lyse, they would not cause harm; capable of digesting the lysosomal membrane
LAMPs and LIMPs
provide protection for the lysosomal membrane from the lysosome's degrative enzyme; highly glycosylated
LAMPs and LIMPs
keep the lysosome from eating itself
AP2
adaptor protein for Clathrin vesicle formation found on the cell surface
Adaptor Proteins
made of four domains, specific to type and tissue (Mickey Mouse ears); can bind receptor tails via tyrosine and dileucine motifs; bind clathrin through their ears;
YTRF
transferrin receptor motif
LDL receptor
uses the "alternate adaptor" - ARH
NPVY
LDL receptor motif
dynamin
GTP hydrolysis here is the vesicle budding/pinching off event
LDL, Insulin, Prolactin
the receptor is recylced back to the cell surface and the ligand is degraded in the lysosome (Pathway 1)
EGF
the receptor accumulates in coated pits only after its binding to ligand and after its dimerization. the receptor is not recycle back to the membrane but degraded in the lysosome; down regulation (Pathway 2)
Diferric Transferrin
both the receptor and the ligand are recycled back to the cells surface
Transcytosis
this process couples endocytosis and exocytosis; antibodies
Diferric Transferrin
iron is stripped off of ligand and ligand dissociates from receptor at the cell surface
Phagocytosis
not a standard endocytic pathway
Lipid Raft
cholesterol rich region of membrane characterized by straightness and rigidity; get the kinks out of unsaturated fatty acids
sphingolipids
thicker parts of membrane are made with a higher concentration of these; cholesterol associates preferably with these
GPI Enriched Early Endosome
internalized tubular structures, enriched in lipid raft components and GP-anchored proteins; from the plasma membrane in a process dependent on Cdc42
Caveolin
integral membrane protein; makes a hairpin loop; coats caveolae
Caveolae Mediated Transcytosis
distinct process found in endothelial cells allowing exchange of serum proteins across the capillary wall
leaky capillaries
result of calveolin knockout in mice
caveolae
only role seems to be transcytosis of albumin and other proteins across endothelia
caveolin
integral membrane protein that also coats endocytic vesicles
soluble hydrolases
transport is usually done by the 46 kDa mannose 6-phosphate receptor (M6P-Rc)
M6P-Rc
uses GGA adaptor proteins to enter into clathrin coated cargo vesicles; recognizes the N-linked glycosylation after ER modifications
activator proteins
transport accomplished by Sortilin
Sortilin
not M6P-Rc; uses GGA adaptor proteins; involved in the tansport of "prosaposin"
prosaposin
activator protein transported by Sortilin
Saposins
assist various lysosomal enzymes in the catabolism of sphingolipids
M6P-Rc and Sortilin
lumenal lysosomal enzymes are brought to the late endosome by these carriers
GGA
Adaptor Proteinl binds receptor, Arf, and clathrin
Lysosomal Membrane Proteins
transport mediated by Adaptor Protein 3 (AP3)
lysosomal membrane glycoproteins
LEP 100, LIMP 1-4, and LAMPs; go to the lysosomes from the Golgi apparatus using adaptor proteins
ear
domain that binds the clathrin in an adaptor protein
AP3
targets lysosomal membrane proteins suchs as LAMPs and LIMPs
Cytoplasmic Protein
have a KFERQ motif which binds to a 73 kDa heat shock protein prior to its delivery to the lysosomes
Retromer
returns the receptors (Sortilin and M6P-Rc) back to the Golgi apparatus; produces tubular buds with unusual morphology
compartment
the nucleolus is an example; not an organelle
nucleolus
structure that generates ribosomal subunits (except 5S)
translation
occurs in the cytoplasm only and not the nucleolus
NPC (Nuclear Pore Complex)
composed of nucleoporins (NUP); central gated channel/tunnel; fuction is transport of molecules across the nuclear membrane
FXFG
motif of nucleoporin NUP62 signaling docking sites for carrier molecules that move in and out of the nucleus
GLFG
motif of nucleoporin NUP98 that assists in RNA trafficking
no repeats
motif of nucleoporin anchoring the NPC into the membrane
perinuclear space
continuous with the ER lumen
SUN and KASH domain proteins
connect nuclear lamina to actin filaments of cytoskeleton
Lamin B
remain associated with membrane during mitosis; found in all cells; isoprenylated
isoprenylation
permanent protein modification on Lamin B generating a "pharmacyl"; covalen modification of C in the CaaX motif
CaaX
motif for prenylation
Lamin Mutations
associated with premature aging and Hutchinson-Gilford Progeria
Barraquer-Simons Syndrome
fat loss disease associated with a defect in Lamin B2
FG
repeats that generate selectivity at the Nuclear Pore Complex
NLS (Nuclear Localization Sequence)
signal that targets to the nucleus
NES (Nuclear Export Signal)
signal that targets to the Cytoplasm
Shuttle Signals
a protein with separate NLS and NES; combines import and export signals
positive
Nuclear Localization Sequences recognizes stretches of these charged amino acids
importin B
binds exclusively to RanGTP
nucleus
concentration of RanGTP high here
cytoplasm
concentration of RanGDP high here
importin B
a.k.a. exportin
FXF
repeats that interact with Ran
RanGTP
higher concentration in the nucleus; binds importin Beta; binds exportin
cancer
trafficking across the Nuclear Pore Complex is faster
nuclear lamins
relatively static; don't move all that much
cycloheximide
protein synthesis inhibitor agent
RNA Pol 1
rRNA (except 5S rRNA)
RNA Pol 2
mRNA (hnRNA), snRNA
RNA Pol 3
5S rRNA, tRNA
introns
bind to splicing factors containing nuclear retention signals
splicing factors
contain nuclear retention signals; bind to introns
5' cap and 3' cap
not necessary for mRNA trafficking
splicing factors
absolutely necessary to get rid of for mRNA trafficking
Crm1
an example of importin B
Crm1
recognizes the NES of the Rev bound to unspliced viral mRNA of HIV-1
Peroxisome
surrounded by a single membrane; plays a role in many detoxification reactions; beta-oxidation of fatty acid chains too long for the mitochondria; involved in the synthesis of specific lipids
post-translation
protein import into the peroxisomal matrix
fully folded
state the protein can be in to be transported into the peroxisomal matrix
energy
requirement of protein import into the peroxisomal matrix
Pex
genes that mediate peroxisomal biogenesis and protein import
Peroxisomal Targeting Sequence
PTS (whats it stand for)
SKL
aka. PTS-1; targetting sequence for the peroxisomal matrix; recognized by Pex5
Pex5 and Pex7
goes into the peroxisomal matrix with cargo attached; then shuffles back and forth; receptor
Pex5
recognizes the SKL motif
Mitochondrial Targeting Sequence
MTS (whats it stand for)
mitochondria
a fully folded protein CANNOT be translocated here; translocation is post-translational but pre-folded
OXA complex
for transmembrane protein insertion into the Inner Membrance
SAM complex
for transmembrane protein insertion into the Outer Membrane
Inner Membrane
mitochondrial sorting to here requires Energy AND a Proton Gradient
Outer Membrane
Mitochondrial sorting to here requires ONLY Energy but no proton gradient
Actin, Microtubules, Intermediate Filaments
Three Components of the Cytoskeleton
F Actin
8 nm in diameter and is highly concentrated at the cell cortex; associated with the plasma membrane; looks like two chains twisted together (the way it polymerizes)
Microtubules
approx. 25 nm in diameter; hollow tube made of tubulin; 13 laterally associated protofilaments, each being a linear polymer. One end of is attached to the centrosome
Intermediate Filaments
10 nm in diameter, a complex assembly of tetramers made from subunits; over 50 types
negative
end that is attached to the centrosome
positive
end that is attached at the periphery
AFs and MTs
polar structures
positive
end of an AF or MT that is growing fast
negative
end of an AF or MT that is growing slowly; growing so slow you can basically forget about it
positive
end of actin that is barbed; usually attached to the periphery
NTP Hydrolysis
happens after incorporation into subunit (i.e. does not grow the filament, or drive polymerization) - hinted as an exam question
NTP Hydrolysis
reduces binding affinity for the D subunit for the neighboring subunits thereby increasing the tendency of the D subunit to dissociate
Lag Phase
in vitro; the period during which nucleation takes place
negative
end of the MT that is attached to the MTOC (the centrosome)
dynamic instability
When a MT is growing it has a cap of GTP tubulin at its positive end because hydrolysis of GTP in the the subunits of the growing end cannot keep up with the rate of polymerization. When polymerization slows down or decreases, the GTP cap is lost and rapid depolymerization takes place
dynamic instability
driven by cycles of GTP hydrolysis
Catastrophe
the transition of a MT end from growing to shrinking state
Rescue
the transition of a MT end from shrinking to growing state
Treadmilling
positive end grows at the same rate that the negative end shrinks when both ends are free in a process called
MAPs
bind to, stabilize, and promote assembly of MTs
Motor Proteins
move along MTs in opposite directions using free energy released from ATP hydrolysis
drugs affecting MTs
stop cell division; possible implications for cancer therapy
drugs affecting MTs
bind to the free tubulin and prevent it from polymerizing causing the Mitotic Spindle to disappear (ex. Colchicine, Vinblasine, and Nocodazole)
Taxol
stabilizes MTs and arrests mitosis like that; 1st drug you get in chemotherapy
Kinesin
positive end directed motor protein
Dynein
negative end directed motor protein
centrosome
site in the cell were MT assembly is initiated. IS a MTOC; MTs attached here via their negative ends
gamma tubulin ring structure
lock-washer; nucleation site for MTs
Kinetochore
MTs that grab the DNA
Polar
MTs that overlap with eachother's positive ends in the middle of the cell; provide the pushing force
Astral
MTS that anchor themselves in the cell periphery; provide the pulling force
NEB
follows phosphorylation of nuclear lamins by m-Cdk; allows for chromosomes to become accessible to the dynamic MTs (search and capture)
centromere
constriction site in the metaphase chromosome; site where the kinetochore assembles
actin myosin contractile ring
accomplish cytokinesis
phalloidin
mushroom toxin that binds actin and causes all of the free monomeric actin in a cell to polymerize and kills the cell```
barbed
where ATP actin gets added to; the growing end
Gelsolin
severs AFs and caps the positive ends; converts viscous, thick AF solution to non-viscous one (in vitro)
Fimbrin
crosslinks AFs to form parallel arrays or bundles
Filamin
gel-forming protein; crosslinks AFs at crosswise intersections forming a viscous gel
CH domain
homologous AF binding domains found on AF crosslinkers (Fimbrin, alpha-actinin, and filamin)
Fimbrin
the AF binding sites are next to each other allowing for tightly packed AFs excluding myosin II
alpha-actinin
the AF binding sites are more widely spaced; found in stress fibers
Filamin
v-shaped dimer with two widely spaced AF binding sites; cross-links the AFs in a loose network with the filaments at seventy degrees to each other
Spectrin
tetramer with two actin binding domains 200 nm apart
rho-GTPases
required for AF cross linking
GDI
GEF inhibitors
Arp2/3
7 protein complex resembling the nucleation site for AFs
WASp
normally assumes an autoinhibited conformation that blocks the A binding site for the Arp 2/3 complex
Cdc42
phosphorylates WASp releasing it from its autoinhibitory state and allowing for the binding of the Arp2/3 complex forming the AF nucleation site
VI
myosin that travels towards the negative end
I
mysosin that does not dimerize
II
myosin that forms antiparallel filaments through aggregation of coiled-coil tails; allows for the muscle cell to slide groups of oppositely oriented AF past each other