Ch 15 - Intracellular Compartments and Protein Transport

- membrane-enclosed organelles - protein sorting - vesicular transport - secretory pathways - endocytic pathways

membrane-bound organelles

surrounded by the cytosol, which is enclosed by the plasma membrane

• occupy nearly 50% of cell volume

• allow spatial separation of different cellular functions

cytosol function

contains many metabolic pathways; protein synthesis; the cytoskeleton

nucleus function

contains main genome; DNA and RNA synthesis

endoplasmic reticulum (ER) function

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

golgi apparatus function

modification, sorting, and packaging of proteins and lipids for either secretion or delivery to another organelle

lysosomes function

intracellular degradation

endosomes function

sorting of endocytosed material

mitochondria function

ATP synthesis by oxidative phosphorylation

chloroplasts (in photosynthetic cells) function

ATP synthesis and carbon fixation by photosynthesis

peroxisomes function

oxidative breakdown of toxic molecules

cytosol percentage of total cell volume (highest)

55%

likely originated by invagination of the plasma membrane

nuclear envelope, membranes of ER, golgi apparatus, endosomes and lysosomes

vesicular traffic

how organelles (w/ exception of nucleus) communicate extensively with one another and with the outside of the cell

likely evolved from bacteria that were engulfed by primitive eukaryotic cells

• remain isolated from the vesicular traffic

mitochondria and chloroplasts

signal sequence of protein

directs the protein to a particular organelle

cytosol

where synthesis of virtually all proteins begins

remain in the cytosol

proteins that lack a signal sequence

into the nucleus, mitochondria, chloroplasts, peroxisomes and the ER

where different signal sequences direct proteins

depends on the organelle

the mechanism by which a protein is transported into an organelle

first mechanism of transport

transport of folded proteins into the nucleus through nuclear pores

second mechanism of transport

transport of unfolded proteins into the ER, mitochondria or chloroplasts across their membranes by protein translocators

third mechanism of transport

transport of proteins in transport vesicles that pinch off from the ER and fuse with a compartment of the endomembrane system

common themes of mechanisms of protein import into organelles

• sequence-specific signal on the “cargo” protein

• signal sequence is recognized by receptor proteins

• directional movement by transport machinery requires energy

nuclear import

large pore, competent for fully folded proteins/complexes. GTP hydrolysis provides energy

mitochondria/chloroplasts

narrow translocation channel; proteins are unfolded and pulled into organelles by chaperone proteins (which hydrolyze ATP for energy)

ER

proteins enter as they are being synthesized

signal sequences

• necessary and sufficient to direct a protein to a particular destination

• typically 15-60 amino acids long; exact sequence can vary

• hydrophobicity or the order of charged amino acids are more important than the exact sequence

• often (but not always) removed from the protein once it has been sorted

deleting a signal sequence from an ER

converts it into a cytosolic protein

adding an ER signal sequence to a cytosolic protein

directs it to the ER

the nuclear envelope

surrounds the nucleus, and consists of an inner membrane and an outer membrane

• outer membrane = continuous w/ ER

  • inner membrane = contains proteins that act as binding sites for chromosomes and the nuclear lamina

what transport across the nuclear envelope occurs through

nuclear pores

the nuclear pore complex

• acts as a selective gate

• large structure composed of ~30 different proteins

• proteins that line it are unstructured and form a mesh that fills the pore, preventing passage of large molecules

how proteins enter the nucleus

in their mature, fully folded state

nuclear localization signal (NLS)

signal sequence that directs transport into the nucleus

nuclear import receptor

cytosolic protein that NLS is recognized and bound by

• interacts w/ fibrils that extend from nuclear pore complexes

• disrupts the protein mesh that fills the pore and opens a passageway into the nucleus

Ran

a small monomeric GTPase that exists in two conformations: one bound to GTP and one bound to GDP

Ran-GTP

is present in high concentrations in the nucleus

Ran-GDP

is present in high concentrations in the cytosol

GTP hydrolysis

facilitates nuclear transport

in the nucleus, Ran-GTP

binds a nuclear import receptor, allowing the prospective nuclear protein to be released

the import receptor, bound to Ran-GTP

returns to the cytosol

hydrolysis of GTP causes

Ran-GDP to release the import receptor, freeing it to bind a new prospective nuclear protein

proteins must unfold

in order to enter mitochondria or chloroplasts

step 1

protein synthesis and folding in the cytosol

step 2

binding of signal sequence to an import receptor on mitochondrial surface

step 3

simultaneous transport across the outer and inner membranes by protein translocators. the protein is unfolded during the transfer process

step 4

cleavage of the signal sequence

mitochondrial chaperone proteins

help pull in and refold proteins (use energy from ATP hydrolysis)

proteins imported into peroxisomes

do not need to unfold, are imported via a related mechanism and also by vesicular transport

ER is the entry point for proteins destined for …

the ER, golgi apparatus, endosomes, lysosomes, and proteins destined for the cell surface

protein will not re-enter the cytosol once it is inside ___________ or embedded in the ______________

the ER lumen, ER membrane

transport vesicles

how proteins are transported to their destination

hydrophobic ER signal sequence

direct proteins that enter the ER while being synthesized

soluble proteins

translocated across the membrane into the ER lumen

transmembrane proteins

partly translocated across the membrane and remain embedded in it

create the rough ER

membrane-bound ribosomes

ribosome synthesizing the protein

attaches to the ER membrane to initiate transfer

structurally identical

membrane-bound and free (cytosolic) ribosomes

polyribosome

when many ribosomes bind the same mRNA molecule

signal recognition particle (SRP)

binds both the ribosome and the ER signal sequence as it emerges from the ribosome

• protein synthesis slows down until this binds to its receptor in the ER membrane

• once bound, it is released, the ribosome is passed to a protein translocator and protein synthesis resumes

soluble proteins made in the ER

released into the ER lumen

cleaved signal

released from the protein translocator into the lipid bilayer and degraded

N-terminal ER signal sequence

initiates transfer into the ER

stop-transfer sequence

halts the transfer

• located further along the polypeptide chain

n-terminal signal sequence is cleaved

stop-transfer sequence remains in the bilayer, forming an alpha-helix that spans the membrane

the protein has a defined and permanent orientation

N-terminus in the ER lumen & C-terminus in the cytosol

start-transfer sequence

an internal signal sequence in some proteins

• initiates protein transfer into the ER

vesicular transport

allows material to exit the cell (exocytosis) and enter the cell (endocytosis)

• continual budding and fusion of transport vesicles enables transport from the ER to the golgi apparatus, and from the golgi to other compartments (this is energetically unfavorable)

is highly selective

secretory pathways

ER → golgi apparatus → cell surface

• at golgi, a side branch leads through endosomes to lysosomes

Quality control mechanism: proteins are checked for proper folding and assembly so that only correctly build proteins make it to the cell surface

misfolded proteins and incorrect assemblies are degraded inside the cell

endocytic pathway

plasma membrane → endosomes → lysosomes

• allows ingestion and degradation of extracellular molecules

vesicle budding

driven by the assembly of a protein coat

clathrin-coated pit

clathrin molecules assemble into a basketlike network on the cytosolic side of the membrane

clathrin

protein that makes up protein coat

step 1 of mechanism of budding of a clathrin-coated vesicle

cargo receptors bind molecules selected for transport

step 2 of mechanism of budding of a clathrin-coated vesicle

adaptins capture cargo receptors and bind clathrin. clathrin assembles into a basketlike array, forming a clathrin-coated pit

step 3 of mechanism of budding of a clathrin-coated vesicle

dynamin (a GTPase) assembles as a ring around the neck of a clathrin-coated pit, hydrolyzes GTP to help pinch off the vesicle

step 4 of mechanism of budding of a clathrin-coated vesicle

following budding, the vesicle sheds its protein coat (ie clathrin and adaptins), allowing direct interaction with the membrane of its target compartment for fusion. uncoating of clathrin requires energy from ATP hydrolysis

Rab proteins (GTPases) on the vesicle surface

are recognized by tethering proteins on the target membrane

matching Rab and tethering proteins

ensures that transport vesicles fuse only with the correct membrane

vesicle docking depends on :

tethers, Rab proteins and SNAREs

SNARE proteins

drive vesicle fusion

SNARES on the vesicle

(v-SNAREs) bind complementary t-SNAREs)

SNAREs on the target membrane

t-SNAREs

during vesicle fusion,

complementary SNAREs wind around one another, pulling the vesicle’s membrane close to the target membrane and displacing water molecules

mediated by transport vesicles

movement between compartments

energetically unfavorable processes

vesicle budding, scission and fusion

budding

driven by protein coat assembly (clathrin, COP)

scission

(pinching off from membrane) is driven by assembly and GTPase activity of Dynamin

fusion

driven by v-SNARE/t-SNARE winding

cargo selection

involves specific cargo protein, a receptor for the cargo, and adaptins that link the receptor to the clathrin coat, and adaptins that link the receptors to the clathrin coat

provide selective vesicle docking

rabs/tethering proteins

covalently modified in the ER

most proteins

disulfide bond formation

catalyzed by an enzyme in the ER lumen

disulfide bonds

help stabilize the structure of secreted proteins

glycosylate in the ER

many proteins

a branched oligosaccaride containing 14 sugars

transferred from a lipid (called dolichol) to the side chain of an asparagine amino acid

N-linked

oligosaccharides linked to an asparagine side chain

subsequent modification of the oligosaccharide

begins in the ER and continues to the golgi

glycosylation

• helps protect a protein from degradation by preventing binding of proteases

• can help mediate binding to chaperone proteins

• this ensures that the protein will be retained in the ER until it is properly folded → quality control mechanism

oligosaccharides

serves as a transport signal for packaging the protein into an appropriate transport vesicle

• on cell surface, can function in cell-cell recognition