ANAT262 - Post Midterm Stuff

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McGill Winter 2012

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

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