mbb 201 lec 15-17

what is the purpose of membrane enclosed organelles

they are important for creating distinct environments with different metabolic functions

functions of cytosol

they contain many metabolic pathways; protein synthesis, cytoskeleton

nucleus main function

contains main genome; DNA/RNA synthesis

endoplasmic reticulum (ER) main function

synthesis of most lipids, synthesis of proteins for distribution to many organelles and plasma membrane

golgi apparatus main function

modificaiton, sorting, packaging of proteins + lipids for secretion or delivery to other organelles

lysosome main function

intracellular degradation

endosomes main function

sorting of endocytosed material

mitochondria main function

atp synthesis by oxidative phosphorylation

chloroplasts in plants main function

atp synthesis and carbon fixation by photosynthesis

main function of peroxisome

oxidation of toxic molecules

evolution of membrane enclosed organelles

1) nuclear membranes and membranes of endomembrane system (er, golgi, peroxisomes, endosome, lysosomes) my have arisen through invagination of plasma membrane

2) interiors of endomembrane system communicate with each other exclusively

protein sorting

almost all proteins begin synthesis in cytosol and are transported through 3 mechanisms:

1. transport through nuclear pore (to nucleus)

2. transport across organelle membranes (proteins translocator)

3. transport by vesicles (endomembrane system)

they are directed by the signal sequence created from the amino acid

signal sequencing

usually 15-60AA long and are necessary to direct a protein to a particular destination. they are removed often after the finished protein has been sorted

transport through nuclear pore

nucleus has nuclear envelope with two membranes (inner and outer, outer is connected w/ ER) and it has pores to allow the passes of molecules in and out

nuclear pore complex

forms a gate in which molecules can enter or leave present in both inner and outer membranes. composed of 30 or so proteins which are disordered and unstructured. they create a fibril mesh that fills the center of the channel and prevents the passage of molecules through it. only small, water solube molecules can pass non-selectively.

protein transport through nuclear pore

cytosoclic proteins that are for the nucleus must contain a nuclear localization signal NLS. it is recognized by nuclear import receptors which helps direct the protein to the pore by interacting with cytosolic fibrils and through the pore by disrupting the interactions between nuclear fibrils. they are fully folded when being transported.

what are cytosolic proteins

Cytosolic proteins are proteins synthesized on free ribosomes and located in the cytosol, the fluid component of the cytoplasm.

gtp hydrolysis drives nuclear transport

nuclear import receptors are returned to the cytosol but requires GTPase (aka Ran). binding of ran-gtp causes teh dissociation of imported proteins from receptor. the receptr bound to ran-gtp can be transported back into the cytosol where gtp is hydrolyzed to GDP. hydrolysis frees ran-gdp from receptor so it can bind with another NLS

nuclear export

similar to process of exporting proteins and also depends on ran-gtp.

protein sorting in mitochondria and chloroplast

has a signal sequence at N-terminus to allow for their import. proteins are unfolded when transported by translocator →signal sequence is removed after arrival and chaperone proteins help proteins fold again

protein sorting in endoplasmic reticulum

have ER signal sequence.

1. soluble proteins in lumen of ER: found in lumen of organelle in endomembrane system or get secreted out of cell

2. transmembrane protein: end up in the membranes of the organelles of the endomembrane system or on the plasma membrane

soluble proteins and ER

soluble proteins made on the ER are released into teh ER lumen. most protein that enter the ER are threaded across the ER membrane before the polypeptide chain is fully synthesized.

ribosomes synthesizing proteins

1. membrane bound ribosomes: attached to cytosolic side of ER

2. Free Ribosomes: not attached to any membrane, identical to Membrane bound

3. Polyribosomes: many ribosomes bound to one mRNA molecule

directing the ribosome to the ER

two protein components help guide ribosome to ER

works by guiding ER signal to Er membrane

1. signal recognition particle (SRP) presented in cytosol and binds to ER signal sequence and ribosome

2. SRP receptor: embedded in ER membrane. bind to SRP and passes ribosome to a protein translocator. SRP is released.

protein synthesis occurs passing the protein through the channel in the protein translocator

Soluble proteins cross the ER membrane and enter the lumen via the translocator channel

ER signal sequence causes the opening of the channel. signal sequence remains bound to the channel as the rest of the protein is threaded through. Once the C-terminus has passed through, the signal sequence is removed by a signal peptidase on luminal side of ER and protein is released into lumen. cleaved signal sequences

transmembrane proteins in the ER

Some of the polypeptide chain must be translocated completely, while other parts must be fixed in the membrane. for a single-pass transmembrane protein, translocation is initiated by start-transfer sequence. translocation continues until a stop-transfer sequence is reached. stop-transfer forms alpha helix and remains embedded din membrane. orientation of N and C will not change. sometimes the start-transfer seq is internal and does not get removed like n-terminus.

enter into ER lumen or membrane is usually only the first step on a pathway to another destination

the end goal is usually the golgi apparatus where they are modified and sorted for shipment to other places.

vesicular transport

the continual budding and fusion of transport vesicles from ER → golgi → other compartments of endomembrane system

the movement of material between organelles in the eukaryotic cell via membrane-enclosed vesicles. allows for transport of both soluble and transmembrane proteins to various parts of the cell including the endomembrane system and plasma membrane. each organelle must maintain its own distinct identity

vesicle budding and coated vesicles

driven by assembly of protein coat.

coated: membeanr enclosed sacs that wear a distinctive layer of proteins on its cytosolic surface to help shape the membrane into a bud and captures molecules for onward transport.

Clathrin-coated vesicles and COP-coated vesicles

Clathrin-coated vesicles: Found budding from the Golgi to endosomes as well as from the plasma membrane on the inward endocytic pathway

COP-coated vesicles: Found in vesicles between the ER and Golgi, as well as from one part of the Golgi to another part of the Golgi

clathrin coated vesicle

vesicle begins as clathrin-coated pit

clathrin is a protein that creates a basker like network on the cytosolic surface of the membrane, helps shape membrane to a vesicle. small gtp-binding protein dynamin functions to pinch off vesicle and assembles a ring around the neck of each invaginated coat pit.

adaptins secure clathrins to vesicle and help select cargo molecules by binding to cargo receptors. appropriate cargo proteins will have transport signals that can be recognized by the cargo receptors.

Clathrin-Coated Vesicles transport selected cargo molecules

different adaptin for different cargo ( they recognize different cargo receptors). once budding is complete the coat proteins are removed and the vesicle and fuse w/ target membrane

recognition, docking, and fusion of vesicles with its target organelle occurs.

markers must be recognized by complementary receptors on the appropriate target membrane. identification is based on diverse GTPase (rab protein) and transmembrane protein v-snare and t-snare

recognition of vesicle: tethering via Rab protein

tethering: rab protein are recognized and bounded by tethering proteins found on target membrane bringing two into close proximity

Recognition of Vesicles: Docking via the SNAREs

Docking: The v-SNARE on the vesicle interact with complementary t-SNAREs (“t” for target) which firmly docks the vesicle in place

Recognition of Vesicles: two lipid bilayers intermix

fusion: the vescivle fuse w/ the target membrane and cargo protein is delivedto the interior of the organelle or secreted if at the plasma membrane. fusion of membranes is energetically unfavourable. fusion occurs when v-snares and t-snares wrap tightly around each other, whinching the vesicles closer to the membrane such tha two membranes are close enough for their lipids to intermix

modifying protein in the ER

formation of disulfide bonds: covalent bonds that link paris of cysteine side chains and stablize protein structures

glycosylation: covalent attachments of short branched oligosaccharides (glycoproteins). protects from degredation, hold protein in the er, recognition by proteins for packaging or cell-cell interactions, glycosylation is rare on the cytosolic side

protein glycosylation in ER

oligosaccharides are not added one at a time but all together. 14 sugar oligosaccharide is originally attached to a specialized lipid dolichol in the ER membrane. then transferred onto the amino group of the asparagine side change as the peptide is translocated. because they are attached to an amino, they are said to be N-linked

exit from the ER is controlled

some proteins stay in ER andwill contain approprate retiontion signal sequence while if they escape, they will be recognized by receptors and sent back to ER.

exit from the er is highly selective: must be properly folded

misfolded or multimeric proteins that do not assemble properly are retained in the ER by the binding of chaperone proteins (prevent misfolding from aggregating). N-glycosylation is a sensor for whether a protein is folded properly. if it still fails ,it will be exported to cytosol where it will be degraded.

unfolded protein response

if too many unfolded proteins accumulate in ER, then the unfolded protein response (UPR) is triggered. more chaperones and quality control relaetd proteins are produced and inhibit protein synthesis. size of ER can be expanded to cope w/ the load but if it exceeds then it can be programmed to die.

further protein modification in the golgi

• cisternae: flattened membrane enclosed sacks (cis = faces towards ER, trans faces plasma membrane, medial cisterna is in the middle)

• enter from cis golgi, protein exit from trans, and modified in golgi.

• trans is the main sorting station

secretory proteins are released from the cell by exocytosis

exocytosis: vesicles from golgi fuse w/ plasma membrane

constitutive exocytosis pathway: supplied the plasma membrane w/ lipids and proteins. some proteins are secreted doesn’t need signal sequence other than ER enterance.

regulated exocytosis pathway: only operates in cells specialized for sections. hormones, mucus, digestive enzumes, etc. proteins ae sorted and packed in trans golgi which has conditions that cause proteins to aggregate (acidic pH and high Ca2+). stored in secretory vesicles waiting for a signal. aggregation allows secretory proteins to be at very high concentrations

endocytosis/endocytic pathways

The uptake of material through the invagination of the plasma membrane. Can be broken down into two types based on size:

1. Phagocytosis: involves the ingestion of large particles. Mainly performed by specialized phagocytic cells

2. Pinocytosis: ingestion of fluid and molecules via small vesicles. Performed by all cells. Macrophages removes the equivalent to 100% of its plasma membrane every 0.5 hours!

phagocytosis

uptake for food and defense against infections. aft particle is engulfed, they are enclosed in vesicles called phagosomes (fused w. lysosomes, digesting engulfed partical).

pinocytosis and receptor mediated endocytosis

occurs continuously, plasma membrane forms pinocytic vesicle and is mainly carried by clathrin-coated vesicles that pinch off and fuse with endosomes. indiscriminate, they just trap whatever.

receptor mediated endocytosis: selective uptake of macromolecules using specific receptors.

endosomes: sorting station for endocytic pathway

endolytic vesicles deliver material to and are sorted by endosomes. some are near plasma membrane mature into late endosomes by fusing with one another and are found near nucleus. endosomes maintain an acidic environment w/proton pump possible paths:

• Recycling: returned to the PM

• Degradation: sent to lysosomes

• Transcytosis: move to a different domain of the PM

lysosome

principal site of intracellular digestion. Lysosomes are acidic and contain many hydrolytic enzymes involved in the degradation of macromolecules. Lysosomal membrane proteins are highly glycosylated on the luminal side – protects from degradation. Contains a proton pump as well as transporters for macromolecule subunits to enter the cytosol. Lysosome destined proteins receive a mannose 6-phosphate tag in the ER and Golgi

autophagy

process by which a cell digest molecules and organelles that are damaged (eats itself). organelle is enclosed by a double membrane, creating an autophagosome which then fuses with a lysosome for destruction

signal transduction

cell communication -

cell communication - endocrine

cell communication - paracrine

cell communication - synaptic

cell communication - contact-dependent

same signal, different response

multiple extracellular signals can dictate how a cell behaves

extracellular signal molecule binds either to cell surface receptors or intracellular receptors

can fall into two categories:

1. molecules that do not cross the plasma membrane and bind to surface receptors (large and hydrophilic)

2. molecules that cross plasma membrane and enter cytosol and bind to intracellular receptors (small, hydrophobic)

steroid hormones

rely on intracellular receptors. they are hydrophobic molecules that cross the plasma membrane. bind to nuclear receptors that when bound to ligand can enter the nucleus and imitate transcription.

nitric oxide

cell surface receptors

cell signaling pathways

molecular switches

signaling by protein phosphorylation

phosphorylation cascades

GTP-binding proteins

monomeric GTPases

Cell-surface receptors

All cell-surface receptors proteins bind to an extracellular signal molecule and transduce its message into one or more intracellular signaling molecule that alter cell’s behavior.

Three major classes:

1. Ion-channel-coupled receptors

2. G-protein-coupled receptors

3. Enzyme-coupled receptors

ion channel-coupled receptors

g-protein coupled receptors

enzyme coupled receptors

g-protein coupled receptors (GPRCs)

Activation of a GPCR

switching off

some g-proteins directly regulate ion channels

many g-proteins activate membrane bound enzymes that produce smaller messenger molecules

cAMP signaling pathway can activate enzymes and turn on genes

cAMP glycogen breakdown

cAMP slow responses

inositol phospholipid pathway

Phospholipase C

Calmodulin

Enzyme-Coupled Receptors

Receptor Tyrosine Kinases:

Most RTKs activate the monomeric GTPase Ras

Ras activates a phosphorylation cascard: MAP-kinase signaling

Ras and cancer

Some RTKs create lipid docking sites

Activated Akt promotes cell survival and cell growth