Class 14

Intracellular Compartments and Protein Transportn

proteolytic enzymes

if break cells apart and mix contents in test tube, chemical chaos results and enzymes are degraded by cells own ____ _____

separate compartments

need to separate processes that occur simultaneously into ____ _____

1st strategy→ aggregate different enzymes required for a particular sequence of reactions into a multicomponent complex

• synthesis of DNA and RNA

• assembly of ribosomes

2nd strategy→ most highly developed in eukaryotes— confine different metabolic processes and proteins required into membrane-enclosed compartments

membrane-enclosed organelles

eukaryotic cells contain a basic set of ___ ___ ___

• nucleus

• endoplasmic reticulum

• Golgi apparatus

• lysosomes

• endosomes

• peroxisomes

• mitochondria

on average, ___ ___ ___ occupy nearly half the volume of eukaryotic cell

evolved in different ways

except for mitochondria and chloroplasts, ___ ___ ___ probably evolved in stages, most likely by invagination of plasma membrane

nucleus

double membrane, nuclear envelope, perforated by nuclear pores

outer nuclear membrane continuous with ER membrane

contains main genome

endoplasmic reticulum

ER

system of interconnected membranous sacs and tubes that often extend throughout most of cell

major site of synthesis of new membranes in cell

smooth and rough ER

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

rough ER

synthesizes most of lipids

large areas of ER have ribosomes attached on cytosolic surface

ribosomes are actively synthesizing proteins that are

• inserted into ER membrane

• delivered to ER interior (lumen)

smooth ER

lacks ribosomes

scanty in most cells but highly developed for certain functions in certain cells

in certain cells, ___ ___ is highly developed for specific functions

• steroid hormone synthesis in some endocrine cells of adrenal gland

• variety of organic molecules, including alcohol, are detoxified in liver cells

• sequesters Ca²⁺ from cytosol; release and reuptake of Ca²⁺ by ER is involved in muscle contraction and responses to extracellular signals

Golgi Apparatus

usually situated near nucleus

received proteins and lipids from ER

modifies proteins and lipid received from ER

dispatches them to other destinations in cell

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

lysosomes

sites of intracellular digestion

small sacs of digestive enzymes

degrade worn-out organelles, macromolecules, and particles taken into cell by endocytosis

endosomes

series of compartments endocytosed materials first pass through on way to lysosomes

sort ingested molecules-

recycle some of ingested molecules back into plasma membrane

sort endocytosed material

peroxisomes

small organelles that contain oxidative enzymes that break down lipids and destroy toxic molecules

use O₂ to perform oxidation reactions

produce H₂O₂ and use it oxidation reactions

when excess H₂O₂ accumulates in cell, catalase enzyme in ______ converts it to H₂O

• 2H₂O₂→ 2H₂O + O₂

oxidative breakdown of toxic molecules

mitochondria

surrounded by double membrane

sites of oxidative phosphorylation

contain internal membranes highly specialized for ATP production

contain mtDNA and ribosomes

ATP synthesis by oxidative phosphorylation

internal membranes

in eukaryotic cells, ____ ___ create enclosed compartments that separate different metabolic processes

in liver enzymes

small, black granules between compartments→ aggregates pf glycogen and enzymes that control its synthesis and breakdown

cell lining from intestine

contains basic set of membrane-enclosed organelles found in most animal cells

distinct compartments separated from cytosol by at least one selectively permeable membrane

ER, Golgi apparatus, mitochondria

many membrane-bound organelles including ____,____, and ____ are positioned in cell by attachment to cytoskeleton, especially to microtubules

cytoskeletal filaments

provide tracks for moving organelles around and for directing traffic of vesicles between organelles

movement of organelles along _____ ____ driven by motor proteins that use energy of ATP hydrolysis to propel them

membranes of organelles

enormous in size

• in typical mammalian cell, area of ER membrane 20-30X area of plasma membrane

• plasma membrane→ minor membrane in most eukaryotic cells

archaea and bacteria

can get by with just a plasma membrane because they are so small

surface area : volume ratio → sufficient

eukaryotic cells

volumes 1000-10,000X higher than prokaryotes

plasma membrane does not provide enough functional membrane for such a high cellular volume

internal membranes→ higher surface area : volume ratio

invagination of plasma membrane

except for mitochondria and chloroplasts, membrane-enclosed organelles probably evolved in stages, most likely by ___ ___ ___ ____

ex.

• nuclear and ER membranes

modern bacteria and archaea

in these, a single DNA molecule is typically attached to plasma membrane

possible that, in a very ancient anaerobic archaeon, plasma membrane with its attached DNA could have invaginated and, in subsequent generations, formed a two-layered envelope of membrane completely surrounding the DNA

this envelope is presumed to have eventually pinched off completely from plasma membrane, ultimately producing a nuclear compartment penetrated by channels called nuclear pores, which enable communication with cytosol

other portions of invaginated membrane may have formed in the ER, which would explain why space between inner and outer nuclear membranes is continuous with ER lumen

endomembrane system and evolution

two points consistent with proposed evolutionary origin of endomembrane system based on invagination of plasma membrane

• interiors of organelles are treated by cell in many ways as extracellular

• nucleus has two membranes

endomembrane system components

ER, Golgi apparatus, peroxisomes, endosomes, lysosomes

interiors of these organelles communicate extensively with one another and with outside of cell by means of small vesicles that bud off from one organelle and fuse with another

protein sorting

before eukaryotic cell divides, it must duplicate its membrane-enclosed organelles

as cell grows, membrane-enclosed organelles get bigger and organelles divide

during cell division, organelles are distributed between two daughter cells

directing newly-synthesized proteins to correct organelle is necessary for cell to function, grow and divide

proteins

delivered directly from cytosol for

• mitochondria

• chloroplasts

• interior of nucleus

• ER

• peroxisomes (bulk of digestive enzymes from cytosol)

proteins and lipids

delivered indirectly via ER for

• Golgi apparatus

• lysosomes

• endosomes

• inner nuclear membrane

• peroxisomes

proteins enter ER

directly from cytosol

• some retained in ER but

• most transported to Golgi by vesicles and then onward to plasma membrane or other organelles

ER itself→ also major site of protein and lipid synthesis

address labels

contained in amino acid sequences

correct address

once at ____ ___, protein enters membrane or interior lumen of organelle

transport into organelles

there are three mechanisms for protein ___ ___ ___, all of which require energy

virtually all proteins in cell begin synthesis on ribosomes in cytosol

fate of protein synthesized in cytosol depends on its amino acid sequence which contain a sorting signal

if no sorting signal, protein stays in cytosol

sorting signal

directs protein to organelle that needs it

initiation of protein synthesis exception

the few proteins synthesized on ribosomes inside mitochondria and chloroplasts

different sorting signals

direct proteins into nucleus, mitochondria, peroxisomes, and ER

hydrophobic bilayer

hydrophilic, soluble proteins face the problem of having to get across the ____ ____

mechanism 1

cytosol ←→ nucleus

proteins transported through nuclear pores, which penetrate both inner and outer membranes

protein remains folded during transport

nuclear pores

selective gates that actively transport specific macromolecules and also allow free transport of smaller molecules

perforate the double membrane of the nuclear envelope

made up of a complex of ~30 different proteins, each present in multiple copies

RNA molecules, which are synthesized in nucleus and ribosomal subunits, which are assembled in nucleus are exported to cytosol

newly synthesized proteins destined for nucleus are imported through ___ ___

mechanism 2

cytosol→ ER / cytosol→ mitochondria

proteins transported across organelle membrane by protein translocators

usually, transported protein must unfold

mechanism 3

ER→ other compartments of endomembrane system→ other compartments of endomembrane system

proteins transported by transport vesicles which pinch off from one membrane of one compartment and then fuse with second membrane of a second compartment

deliver soluble proteins and also proteins and lipids of vesicle membranes

protein remains folded

signal sequence

direct proteins to correct compartment

typical ___ ___ on a protein is a continuous stretch of amino acid sequence, typically 15-60 amino acids long

often cleaved off finished protein once it has been sorted

recombinant DNA techniques

can be used to change the destination of two proteins

• proteins destined for ER possess an N-terminal signal sequence that directs them to that organelle, whereas those destined to remain in the cytosol lack any such signal sequence

• if the signal sequence is removed from an ER protein and attached to a cytosolic protein, both proteins are reassigned to the expected, inappropriate location

inner nuclear membrane

contains

• some proteins that act as binding sites for chromosomes

• other proteins that provide anchorage for nuclear lamina

nuclear lamina

finely woven meshwork of protein filaments that lines inner face of nuclear membrane and provides support for nuclear envelope

outer nuclear membrane

closely resembles ER membrane with which it is continuous

is penetrated by nuclear pores

nuclear pore complex

forms a gate through which selected macromolecules and larger complexes enter or exit the nucleus

protein fibrils

• protrude from both sides of pore complex

• on nuclear side, they converge to form a basket-like structure

• spacing between the fibrils is wide enough that the fibrils do not obstruct access to the pores

many proteins lining nuclear pore

form a soft, tangled meshwork , like a kelp forest, that fills center of channel and prevents passage of large molecules but allows small water-soluble molecules to pass back and forth

nuclear location signal

must be displayed in order to gain entry to pore

recognized by nuclear import receptors, which interact with the cytosolic fibrils that extend from the rim of the pore

nuclear transport

driven by energy supplied by GTP hydrolysis

nuclear import receptors

recognize nuclear localization signal

help direct newly synthesized protein to pore by interacting with tentacle-like fibrils extending from pore rim into cytosol

___ ___ ___ disrupt interactions between nuclear pore proteins which helps clear a path through pore’s interior meshwork

___ ___ ___ return to cytosol via nuclear pores for reuse

nuclear export receptors

work in a similar way to nuclear import receptors, driving protein and RNA traffic from nucleus to cytosol

export mRNAs from nucleus

recognize nuclear export signals

chaperone proteins

____ ____ inside mitochondria help pull proteins across membranes and fold it once it is inside

subsequent sorting takes place to get protein to correct part of mitochondria

• insertion of transmembrane proteins into inner membrane is guided by signal sequences in protein that start and stop transfer process across membrane

mitochondrial membrane phospholipids

most are thought to be imported from ER, the main site of lipid synthesis in the cell

most mitochondrial proteins

are encoded by nuclear DNA and synthesized in cytosol

usually have a signal sequence at N terminus that allows them to enter mitochondria

are translocated simultaneously across both inner and outer membranes at specialized sites where two membranes closely apposed

each is unfolded as it is transported

signal sequence removed after translocation complete

mitochondrial precursor proteins transport step 1

mitochondrial signal sequence on a mitochondrial precursor protein recognized by receptor in mitochondrial OM

receptor associated with protein translocator which transports signal sequence across OM to intermembrane space

mitochondrial precursor proteins transport step 2

complex of receptor, precursor protein, and translocator then diffuses laterally in OM until signal sequence recognized by a second translocator in IM

mitochondrial precursor proteins transport step 3

together, two translocators transport protein across both OM and IM, unfolding protein in process

mitochondrial precursor proteins transport step 4

signal sequence is cleaved off by a signal peptidase in mitochondrial matrix

proteins enter peroxisomes

____ ____ ____ from both cytosol and ER

peroxisomes

packed with enzymes that

• digest toxins

• synthesize certain phospholipids, including those in myelin sheath surrounding nerve cell axons

mainly get proteins from cytosol by selective transport

peroxisomal protein import signal

short sequence of three amino acids

peroxisomal membrane

has a translocator that helps in protein transport, but proteins do not need to unfold to get into peroxisomes

a few of the proteins embedded in ____ ___ arrive via vesicles that bud off from ER

Zellweger syndrome

peroxisomes clinical implication

born with severe abnormalities in brain, liver, kidneys, and most do not survive past first 6 months

caused by mutations that block peroxisomal protein import, showing that peroxisomes and peroxisomal protein transport is crucial to life

endoplasmic reticulim

most extensive membrane system in eukaryotic cell

• of total membrane in mammalian cell, up to 50-90% surrounds ER lumen

continuous network of flattened sacs, tubules, and associated vesicles that stretches throughout cytoplasm of eukaryotic cell

site of synthesis of proteins destined for

• plasma membrane

• organelles of endomembrane system

• secretion by cell

central role in lipid synthesis

ER cisternae

membrane-bounded sacs

ER lumen

space enclosed in ER cisternae

proteins enter ER

while being synthesized

water-soluble proteins

completely translocated across ER membrane and released into ER lumen

will either be

• secreted

• transported to lumen of other organelle

prospective transmembrane proteins

partly translocated across ER membrane and become embedded in it
will either

• stay in membrane of plasma membrane, or

• one of organelles of endomembrane system

endoplasmic reticulum protein transport

different from transport across membranes of other organelles because transport starts before polypeptide chain has been completely synthesized

ribosome synthesizing protein is attached to ER membrane (RER)

ribosomes in cytosol

two populations

• membrane-bound ribosomes

• free ribosomes

membrane-bound ribosomes

attached to the cytosolic side of ER membrane and outer nuclear membrane making proteins being translocated into ER

free ribosomes

making all the other proteins encoded by nuclear DNA

proteins with ER sequence

are being translocated as they are synthesized so no additional energy needed for transport across ER membrane

elongation of each polypeptide provides thrust needed to push growing chain through ER membrane

polyribosome

as mRNA molecule translated, many ribosomes bind to it, forming a _____

if proteins being synthesized have ER signal sequence, then _______ gets attached to ER membrane

ER signal sequence

8 or more hydrophobic amino acids

directs protein to ER

common pool of ribosomes

is used to synthesize all the proteins encoded by the nuclear genome

• ribosomes that are translating proteins with no ER signal sequence remain free in the cytosol

• ribosomes that are translating proteins containing and ER signal sequence on the growing polypeptide chain will be directed to the ER membrane

• many ribosomes bind to each mRNA molecule, forming a polyribosome

• at the end of each round of protein synthesis, the ribosomal subunits are released and rejoin the common pool in the cytosol

soluble proteins made in ER

are released into the ER lumen

three protein components

help guide ER signal sequences to ER membrane

• signal recognition particle (SRP)

• SRP receptor

• protein translocator

ER signal sequence and SRP

direct a ribosome to the ER membrane

1. SRP in cytosol binds to both the exposed ER signal sequence and the ribosome, therefore slowing protein synthesis by the ribosome

2. SRP-ribosome complex then binds to an SRP receptor in the ER membrane

3. SRP is released, and the ribosome passes from the SRP receptor to a protein translocator in the ER membrane

4. protein synthesis resumes, and the translocator starts to transfer the growing polypeptide across the lipid bilayer

soluble protein to ER lumen

signal sequence

• opens protein translocator

• stays bound to translocator while rest of polypeptide chain threaded through membrane as large loop

• removed by transmembrane signal peptidase

1. protein translocator binds the signal sequence and threads the rest of the polypeptide across the lipid bilayer as a loop

2. at some point during the translocation process, the signal peptide is cleaved from the growing protein by a signal peptidase

3. this cleaved signal sequence is ejected into the bilayer, where it is degraded

4. once protein synthesis is complete, translocated polypeptide is released as a soluble protein into ER lumen

start and stop signals

determine arrangement of a transmembrane protein in lipid bilayer

transmembrane proteins

some proteins made by ribosomes attached to the ER remain embedded in ER membrane as ____ ____

some parts of proteins are completely translocated and other parts stay in lipid bilayer

1. signal sequence at beginning initiates translocation, but then

2. stop-transfer sequence further along polypeptide chain stops transfer process

single-pass transmembrane protein

has an N-terminal signal sequence

• cleaved off by signal peptidase

stop-transfer sequence

• remains in bilayer

both sequences are hydrophobic

N-terminal signal sequence

1. N-terminal ER signal sequence initiates transfer

2. the proteins also contains a second hydrophobic sequence, which acts as a stop-transfer sequence

   1. when this sequence enters the translocator,, the growing polypeptide chain is discharged into the lipid bilayer

3. the N-terminal signal sequence is cleaved off, leaving the transmembrane protein anchored in the membrane

4. protein synthesis of the cytosolic side then continues to completion

double-pass transmembrane protein

has internal signal sequence

• signal sequences not cleaved off→ stay and anchor protein in membrane

• if protein spans membrane >2 times

  • additional pairs of start-and stop-transfer sequences repeat process for each pair

internal signal sequence

not only acts as a start-transfer signal, it also helps to anchor the final protein in the membrane

1. like the N-terminal ER signal sequence, the internal signal sequence is recognized by an SRP, which brings the ribosome to the ER membrane

2. when a stop-transfer sequence enters the protein translocator, the translocator discharges both sequences into the lipid bilayer

3. neither the start-transfer nor the stop-transfer sequence is cleaved off, and the entire polypeptide chain remains anchored in the membrane as a double-pass transmembrane protein