ER
continuous network of membranous tubules and sacs that run throughout the cell
gives rise to golgi, lysosomes, and new cell membrane
rough ER
transitional ER
smooth ER
rough ER
has ribosomes on cytosolic surface
important in protein processing
transitional ER
involved with budding that sends vesicles to the golgi
important in protein processing
smooth ER
no ribosomes attached
involved in lipid metabolism
translocation
process of sending a protein into the ER
some proteins targeted for lumen of ER or to be embedded in its membrane
where does all protein synthesis begin
free ribosomes in the cytosol (unattached to ER)
proteins destined to remain in cytosol complete synthesis on free ribosomes
destination of proteins that complete synthesis on free ribosomes
remain in cytosol nucleus mitochondria chloroplasts peroxisomes
destination of proteins that complete synthesis on membrane bound ribosomes
plasma membrane
secretory vesicles
endosomes –> lysosomes
cotranslational translocation
some proteins headed for lumen of ER enter as they are being made (during translation)
cotranslational translocation process
synthesis begins on a free ribosome in the cytosol
proteins have a unique signal sequence near the N-terminus that is about 20 AA long and contains a stretch of hydrophobic AA
signal recognition particle (SRP) that is a protein-RNA complex recognizes signal sequence and binds to the SS and ribosome which halts translation
mRNA-ribosome-polypeptide-SRP complex binds to protein on ER called SRP receptor (SRP receptor binds SRP and SS on polypeptide binds to protein complex next to SRP receptor called translocon
translocon forms channel into ER lumen
binding of SRP receptor to SRP causes SRP to be released from SS and ribosome allowing translation to resume
growing polypeptide inserted into channel in translocon but the SS is retained within the translocon bound to the wall of the channel
signal peptidase associated with translocon on lumen side and cleaves SS releasing polypeptide into lumen when translation is complete
posttranslational translocation
some ER lumen proteins made on free ribosome then translocated into ER
ER membrane proteins with an N-terminus SS and an internal stop transfer sequence
single pass membrane proteins
translocation proceeds as described in cotranslational translocation but midway through synthesis there is a stop transfer sequence that stops translocation (by altering translocon) so the remainder of polypeptide remains on cytosolic side
stop transfer sequence passes through wall of translocon into phospholipid bilayer
when finished - N-terminus on lumen side and C-terminus on cytosolic side with stop transfer sequence embedded in membrane
ER membrane proteins with internal signal sequence(s) and/or internal stop transfer sequence(s)
single or multiple pass membrane proteins
orientation of single pass membrane proteins may be in either orientation (N-terminus or C-terminus on outside)
some proteins have multiple internal signal sequences and stop transfer sequences which results in multiple pass membrane proteins
protein folding and processing in the ER
chaperones/folding
cleavage
disulfide bridge formation
glycosylation/other modifications
chaperones and folding
polypeptides must be in correct folding patter to function properly
correct folding mediated by chaperones (which are also proteins)
complete polypeptide will assume correct folding spontaneously but before translation is complete it can assume an incorrect pattern or aggregate with other partially complete polypeptides
chaperones in ER and cytosol bind to nascent polypeptide to keep it from interacting with anything else until synthesis is complete
prion
improperly folded protein can be disease causing unit can't be destroyed by heat can interact with other properly folded proteins and turn them into prions ex: mad cow disease
cleavage
many polypeptides have AAs removed after translation
removal of inital methionine
extensive cleavage as with preproinsulin
cleavage process to form insulin
preproinsulin has N-terminal SS which is removed inside Er to make proinsulin
proinsulin becomes insulin when internal AA sequence is removed in ER lumen and 2 polypeptide fragments are joined by disulfide bridges
disulfide bridge formation
protein disulfide isomerase responsible for making and breaking disulfide bridges until most stable configuration is formed
ONLY IN LUMEN
disulfide bridge is a covalent bond between 2 cysteine residues
disulfide bridges only found in proteins that are to be secreted or are exterior membrane proteins because the cytosol contains reducing agents that would break the bonds
glycosylation and other modifications
other chemical modifications occur in the ER lumen
glycosylation: addition of oligosaccharides (carbs)
external membrane proteins glycosylated this way
lipids synthesized in the smooth ER
most membrane lipids including phospholipids, glycolipids, and cholesterol
phsopholipid synthesis
occurs in outer layer of ER membrane bilayer
enzyme flippase moves phospholipids to inner membrane layer after synthesized on outer membrane layer
export from ER
vesicles bud off the ER from transitional ER and carry ER lumen content and ER membrane components to the golgi
vesicles first fuse to ER-golgi intermediate compartment which gradually becomes cis-face cisternae of golgi which gradually becomes trans-face cisternae
from trans-face cisternae vesicles bud off to fuse with cell membrane (secretion and cell membrane formation) or fuse with endosomes/lysosomes
where is golgi most abundant
secreting cells (exocytosis)
clathrin coated vesicles
as vesicles bud off the ER, the region to form a vesicle first becomes coated in protein (ex: clathrin) through complex series of reactions
protein forms in network pattern
binding stimulates budding because it distorts membrane
after vesicle buds off, clathrin is removed
membrane fusion
a vesicle fuses with either cell membrane or another membrane (ex: endosome)
process involves binding of v-SNARES (on vesicle) to t-SNARES (on target membrane)
lysosomes
small membranous sacs containing lysosomal acid hydrolases (powerful hydrolytic enzyme)
lysosomal hydrolases
about 50 kinds
can break down all cellular organic compounds
only work in acidic environment (pH ~5) of lysosome (cell would be okay if one burst because wouldn’t be functional in non-acidic cytosol)
endocytosis
extracellular material brought into a vesicle by this
pinocytosis
small scale endocytosis
involves clathrin-coated endocytic vesicles
endosome formation
small endocytic vesicles fuse with early endosomes (larger vesicle than endocytic vesicles)
membrane recycled to cell membrane and early endosome becomes late endosome
hydrolases in golgi carried by vesicles to late endosome and material brought in from outside by pinocytosis is digested
lysosome formation
late endosomes mature into lysosomes with a high concentration of acid hydrolases
phagocytosis
endocytosis on a large scale
phagocytized material enters cell and a phagosome is formed
lysosome fuses with phagosome and digests phagocytized material
autophagy
old organelles surrounded by ER membrane and sac becomes autophagosome
lysosomes fuse with autophagosome and old organelles are digested
mitochondria
organelles specialized for aerobic respiration
believed to have arisen by endosymbiosis of a prokaryote in an ancestral eukaryotic cell
structure of mitochondria
double membrane with inner membrane folded creating cristae
between 2 membranes is inner membrane space and inside inner membrane is the matrix
outer membrane highly permeable to small molecules due to channels formed by porin proteins
inner membrane impermeable to most ions and small molecules
genetic material of mitochondria
circular DNA (like bacteria) 16 kb long in most animals (plant mitochondrial DNA considerably longer) many copies of circular DNA in each mitochondrion
mitochondrial genes include what
genes for a few of the proteins needed for oxidative phosphorylation (the rest are encoded by nuclear genes)
all mitochondrial rRNA genes (bc they have their own ribosomes)
all mitochondrial tRNA genes
human mitochondrial genome
about 16 kb long
encodes 13 proteins that are embedded in inner mitochondrial membrane and are involved in oxidative phosphorylation
encodes the 16S and 12S rRNA and the 22 tRNAs
mitochondrial genetic code deviates from universal genetic code
may contain other genes that are found in open reading frames hidden in other genes
mitochondrial ribosomes
16S and 12S rRNA
16S - large subunit (39S)
12S - small subunit (28S)
ribosomal proteins
all coded for by nuclear genes and the proteins are transported into the mitochondrion
lower RNA/protein ratio in mitochondria versus E. coli
mitochondrial large subunit
16S rRNA
29S
48 proteins (28 similar to E. coli)
mitochondrial small subunit
12S rRNA
28S
30 proteins
mitochondrial gene inheritance
usually passed to the zygote only via the egg and not the sperm (maternal inheritance)
mitochondrial genes seem to have a higher mutation rate so they’re useful in revealing genetic differences between closely related organisms
multiple copies of each mitochondrial gene in every cell so recovery of mitochondrial genetic material from minute samples is easier than recovery of genetic material
evidence for endosymbiosis of mitochondria
circular chromosome with one origin of replication
formylmethionine used in initiation of protein synthesis
similarity of rRNAs to bacteria
other mitochondrial components
majority of mitochondrial proteins imported from cytosol (at least ~1000 proteins encoded by nuclear genes and imported in)
TOM
protein used in outer membrane transport
TIM
protein used in inner membrane transport
signal cell/signaling cell
cell releasing signal
target cell
cell picking up signal
direct cell-cell signaling
communication via direct connection between cells
seen in some embryonic development
types of signaling by secretion
endocrine signaling
paracrine signaling
autocrine signaling
endocrine signaling
hormones (signal molecules) released from endocrine organ and travels through circulatory system to target organ
endocrine organs
pituitary thyroid parathyroids pancreas adrenal glands gonads many others
paracrine signaling
signal molecules travel to local target organs (not via circulatory system)
ex: neurotransmitters that cross synapse in nervous tissue
autocrine signaling
signaling cell and target cell are the same
signal molecules travel very locally (same cell)
ex: proliferation of T cells of immune system which is induced by antigens released by T cells
steroid hormones
can cross cell membrane of target cells because they’re small and hydrophobic
bind to receptor in cell cytosol then travels through nuclear pore into nucleus and acts on DNA to activate transcription of gene
made from cholesterol
testosterone
estrogen
progesterone (sex steroids made chiefly by the gonads)
corticosteroids (made by adrenal gland)
molecules that act like steroids but aren’t steroids
thyroid hormone
vitamin D
retinoic acid
nitric oxide (NO)
important in certain paracrine signaling pathways
involved in signaling that results in blood vessel dilation
half life of only a few seconds making it paracrine signaler
how is NO synthesized
nitric oxide synthetase (enzyme) uses arginine as substrate to form NO
NO effect process
endothelial cells of vessels synthesize NO in response to neurotransmitters
NO travels and enters nearby smooth muscle cells where it activates guanylyl cyclase which synthesizes cGMP (second messenger molecule)
cGMP relaxes smooth muscle which dilates the vessel
nitroglycerin
used in treating heart disease
converted to NO
releases such large amount of NO that it can have affect even with short half life
neurotransmitters
paracrine signalers move from a neuron to another neuron (or muscle), diffuse across the synaptic cleft, and bind to receptor on the surface of the target hydrophilic so can't cross membrane bind to receptor on membrane which opens ion channels on the target cell initiating nerve impulse acetylcholine dopamine epinephrine serotonin histamine glutamate glycine GABA
epinephrine
neurotransmitter but can also function as a hormone secreted by the adrenal gland (called adrenaline in this case)
peptides
a few to more than 100 AA
can’t cross cell membrane so bind to receptors on membrane
insulin and glucagon (pancreas)
hormones of pituitary (growth hormone and FSH)
eicosanoids
lipids that bind to cell surface receptors (paracrine)
prostaglandins
prostaglandins
some promote inflammation
prostaglandin synthesis
enzyme cyclooxygenase (COX) responsible for synthesis
COX
inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs) like aspirin
COX-1
COX-2
effect of blocking COX-1
gastrointestinal problems
aspirin
blocks both COX-1 and COX-2
g protein coupled receptors
largest family of surface receptors
7-pass membrane proteins
may be as many as 1000 types encoded by human genome
epi and prostaglandins
olfactory and taste (bitter and sweet)
behavioral and mood regulation (nt including serotonin, dopamine, GABA, and glutamate)
GCPR process
when signal molecule binds to GPCR the protein undergoes a conformational change on cytosolic side that causes G protein on cytosolic side to release its GDP and exchange it for GTP
alpha chain of G protein tis released and can interact with adenylyl cyclase to form cAMP from ATP
cAMP (second messenger) then travels within the cell and elicits further responses
intracellular signal transduction
cAMP and others
signal molecules may cause increase in intracellular cAMP (second messenger) concentration by activating adenylyl cyclase
cAMP may then activate protein kinase A
protein kinase phosphorylates proteins and in some cases activates them while in other cases inactivates them
protein kinase can also enter nucleus and activate transcription
exosomes
vesicles released from cells with proteins or RNAs
stages of dividing cells
cell growth DNA replication mitosis cytokinesis G1, S, G2, M
S
synthesis
DNA replication occurs during this phase
G1
cell growth
first phase
M
mitosis
G0
non-dividing cells enter this phase after G1 if certain growth factors aren’t present
restriction point
4 major checkpoints in cell cycle
G1/S
S
G2/M
M
G1/S checkpoint
DNA damage checkpoint
checkpoint near end of G1
cell cycle will halt if DNA has been damaged and need repair
S checkpoint
DNA damage checkpoint
mid-S
cell cycle will also halt here if DNA has damage that needs repair
G2/M checkpoint
DNA damage checkpoint
near end of G2
cell cycle will halt if DNA replication is not complete or if DNA damage is detected
regulated by MPF
M checkpoint
spindle assembly checkpoint
around anaphase
mitosis halted unless chromosomes have properly aligned
colchicine is agent that inhibits spindle fiber assembly so cells will not not divide and stay at this point until inhibitor gone
MPF
maturation promoting factor
cyclin B/Cdk1 complex phosphorylated at one amino acid, dephosphorylated at 2 other amino acids
active form regulates entry into mitosis at G2/M checkpoint
CDK
cyclin dependent kinase
how does MPF regulate entry into mitosis
related to continual synthesis of cyclin, its degradation, a kinase that phosphorylates Cdk1, and a phosphatase that dephosphorylates Cdk1
DNA damage activates separate kinase that results in deactivation of MPF (so cell can’t enter into mitosis)
examples of Cdk1/cyclin B’s action
condensins are activated by phosphorylation by Cdk1/cyclin B
initiates breakdown of nuclear envelope that occurs at outset of prophase
fragmentation of golgi apparatus
spindle formation
condensins
protein complexes that are responsible for chromosome condensation during mitosis and meiosis
nuclear envelope breakdown process
involves phosphorylation of lamins by Cdk1/cyclin B causing their depolymerization
phosphorylation of lamins, nuclear pore complexes, and inner nuclear membrane proteins
p53 protein
tumor suppressor gene on short arm of chromosome 17
when DNA is damaged p53 protein causes other proteins to bind to Cdk thereby inactivating it so cell cycle halts
involved in stimulating DNA repair (and if damaged beyond repair stimulates apoptosis)
if p53 damaged - cells with damaged DNA will continue to divide (possibly leading to cancer)
Li-Fraumeni syndrome
person with only 1 copy of p53 gene
have high chance of developing tumors
fragmentation of golgi apparatus process
phosphorylation of golgi matrix proteins via Cdk1/cyclin B activity
spindle formation process
phosphorylation of centrosome and microtubule-associated proteins via Cdk1/cyclin B activity
genetics of cancer
involves 2 classes of gene alterations that produce cancer
oncogenes
tumor suppressor genes
oncogenes
changes in genes that regulate the proliferation of cells is a prerequisite to becoming a cancer cell
oncogene stimulates cell to divide in an unregulated fashion
ras gene family
how do oncogenes enter cells
tumor virus
mutation(s) that occur to existing cell-cycle genes (proto-oncogenes) - more common!!
types of mutations that turn proto-oncogenes into oncogenes
point mutations translocations deletions duplications gene amplification
tumor suppressor genes
in normal cells these genes are present to inhibit growth of cells containing oncogenes
these genes must be altered or deleted in order for a tumor to become malignant
p53
role of miRNAs in tumor production
increased tumor activity is often associated with loss of some normal miRNA activity
percentage of tumors that arise from mutation to proto-oncogene producing oncogene
80%
ras gene family
most common oncogene family in human cancers
25% of all cancers
50% of colon carcinomas
25% of lung carcinomas
point mutations convert a ras proto-oncogene into an oncogene (changes 1 important AA)
normal ras gene activity
normal ras protein made by proto-oncogene bound to cytosolic face of cell membrane and may be inactive (bound to GDP) or active (bound to GTP)
when growth factor (such as platelet derived growth factor - PDGF or epidermal growth factor - EGF) is recognized by a cell it binds to a target cell membrane receptor and cytosolic face of the receptor is phosphorylated
this results in recruitment of GDP-GTP exchange factor to membrane which converts ras protein into active form
this sets off series of reactions that activate cell division
after activation has occurred, the ras protein hydrolyzes its GtP to GDP and cell division is turned off
mutant ras gene activity
altered ras protein made by oncogene is incapable of hydrolyzing GTP to GDP so cell division is constitutively turned on
percentage of cancers with altered p53 gene
50%
normal p53 gene activity
controls cell cycle, DNA repair, and apoptosis
p53 always made but usually is bound to MDM2 protein which degrades and inactivates it
when DNA is damaged, MDM2 dissociates from p53 making it more stable and turning its activity on
p53 is a transcription factor so if DNA damage is not repaired it results in cell-cycle arrest and apoptosis
how does MDM2 dissociate from p53 when DNA damage present
ATM (protein kinase) stimulated by damage
ATM phosphorylates MDM2 and p53 causing MDM2 to lose its ability to bind to and degrade p53
mutant p53 gene
altered p53 can’t arrest the cell cycle, stimulate DNA repair, and cause apoptosis so cells will survive and will have higher mutation rates
BRCA1 and BRCA2 (common in breast and ovarian cancers) have similar ignoring of cell cycle checkpoints