L16: Protein translocation

nucleus functions

storage and expression of genetic info

endoplasmic reticulum functions

• protein production and modification

• lipid production

• Ca2+ storage

golgi apparatus functions

• protein/lipid modification

• carb production

• sorts proteins and lipids produced by the ER to their destinations

mitochondria functions

aerobic resp

chloroplast functions

photosynthesis

endosomes functions

• uptake extracellular substances into the cell and sort it

• many of these substances are sent to the lysosome for degradation

lysosomes functions

degradation of macromolecules

peroxisomes functions

metabolism using oxidative reactions 

size of nucleus + ER

15 micrometers

nucleus structure

• has a double lipid bilayer

• inner nuclear and outer nuclear membranes

inner nuclear membrane

meshed with nuclear lamina

nuclear lamina

• provides structural support for membrane

• links nucleus to exoskeleton

outer nuclear membrane

continuous with ER membrane

perinuclear space

space between inner and outer membrane that is continuous with ER lumen

smooth ER functions

• lipid metabolism

• Ca2+ storage

• smooth looking surface membrane

why does smooth ER synthesize lipids

for other organelles in cell

Ca2+ storage in smooth ER

• sequesters Ca2+ from the cytoplasm and releases it upon a signal

• used for cellular signalling and muscle contraction

rough ER functions

• site of protein production

• multiple ribosomes attach to its surface as they translate mRNA

• gives it the “rough” appearance

where do the newly synthesized polypeptides go

into the ER lumen

why is protein transport into the ER important

• for transmembrane proteins

• for proteins that need to get transported outwards towards the golgi apparatus

transportation of molecules mechanisms

• secretory pathway

• endocytic pathway

• retreival pathway

• autophagy

transmembrane proteins

• exist in the phospholipid bilayer

• type of this protein defines the function of each membrane

how do transmembrane proteins attach to phospholipid bilayer

uses a hydrophobic anchor that embed inside the hydrophobic core of the phospholipid bilayer

what do transmembrane proteins use as anchor

• single and multi pass alpha helices

• beta-barrels

• other hydrophobic molecules that are post-translationally attached to the protein

• ex. fatty acids

alpha helices as membrane anchor

• long stretch of alpha helix composed of hydrophobic side chains embed inside the lipid bilayer

• 20-30 aa long

single pass proteins

• transmembrane proteins that have a single, alpha-helical anchor

• peptide chain passes through the lipid bilayer only one

multi-pass proteins

transmembrane proteins that have multiple alpha-helical anchors

beta-barrel as membrane anchor

• variety of proteins are made of these

• is a beta-sheet that is rolled into a tube

• outer surface of this is hydrophobic, allowing it to embed in bilayer

beta-barrel sizes

• some are smaller with functionally important residues sticking out

• ex. signal receptors

• some are larger with a hole in the middle for small molecules to pass through

• ex. transporters

what could happen to proteins after translation

• protein folding

• post-translational modification

• protein trafficking

• protein degradation

protein folding

• proteins dont fold on their own

• chaperone proteins help other proteins fold properly

post translational modification

• cleavage of the first Met and signal sequences

• addition of cofactors, phosphorylation

protein trafficking

• transporting proteins to their destinations

• occurs in bacteria and eukaryotes

protein degradation

damanged/misfolded proteins get degraded

3 modes of protein transportation in eukaryotes

• gated transport

• protein translocation

• vesicular transport

gated transport

• transport large, already folded molecules through huge pores

• allows passive diffusion of small molecules

• exclusive for transport between nucleus and cytosol

protein translocation

• transport unfolded proteins through the membrane

• assisted by transmembrane proteins

• does not allow diffusion of small molecules

• enters plastids, peroxisomes, mitochondria and the ER

vesicular transport

• uses a membrane enclosed vesicle to ferry molecules

• used by anything past the ER

draw the order of organelles in cells

slide 19

proteins that function in the nucleus

• DNA poly

• RNA poly

• RNA processing proteins

• histones

• transcriptional regulators

macromolecules made in the nucleus but function in the cytoplasm

• mRNA

• ribosome subunits

• tRNA

nuclear pore complex

• NPC

• multi protein structure

• resides in the nuclear membrane

• creates a large pore on its surface

• controls selective transportation between nucleus and cytoplasm

NPC size

125 megadalton in vertebrates

what are NPCs made out of

30 different kinds of nucleoporins in huge quantities

NPC structure

• ring like structure with 8 fold symmetry

• scaffold nucleoporins

• channel nucleoporins

• membrane ring proteins

• cytoplasmic filaments

• nuclear basket

scaffold nucleoporins

• make outer portion of the NPC ring

• defines the shape of the structure

channel nucleoporins

• makes the inner portion of the NPC ring

• constitutes the center channel

membrane ring proteins

• outermost edge of the NPC ring

• transmembrane proteins that anchor NPC to the nuclear membrane

cytoplasmic filaments

• 8 long filaments that stick out on the cytoplasmic side of the NPC

• binding site for nuclear import and export receptors

nuclear basket

• 8 filaments on the nuclear side of the NPC

• constricted at their ends

• create a basket shape

• binding site for nuclear import and export receptors

how is NPC a molecular sieve

• central pore is filled with nucleoporins domains that are rich in G and F

• molecules smaller than 5kDa diffuses freely

• molecules larger than 40kDa cannot pass through diffusion

• must be actively transported with the help of nuclear importer and exporters

signal sequences

• conserved aa sequences coded on proteins that tag them to be transported to their corressponding organelle

• known as ‘cargo’

2 types of signal sequences

• nuclear localization signal (NLS)

• nuclear export signal (NES)

• nuclear import and export receptors bind to proteins with these and guide them to the correct location

Nuclear localization signal

• signal sequence for nuclear import

• patch of basic amino acids

nuclear export signal

• signal sequence for export

• hydrophobic aa every 3-4 nucleotides

nuclear export receptor

• mature mRNA get bound by these and get taken into the cytoplasm from the nucleus

• ex. mRNA

• interact with the FG rich repeats inside the NPC channel

• facilitates the receptors to move through the pore to the other side

nuclear import receptor

• binds to the NLS and imports the proteins into the nucleus from the cytoplasm

• interact with the FG rich repeats inside the NPC channel

• facilitates the receptors to move through the pore to the other side

what is receptor-mediated nuclear transport powered by

Ran-GTP

Ran

• protein

• shuttled between nucleus and cytoplasm

• gets co-transported with nuclear import and export factors while they transit through the NPC

what is Ran bound to

GTP or GDP depending on its location

Ran in cytoplasm

• Ran-GTPase-activating protein (Ran-GAP) hydrolyzes Ran-GTP

• produces Ran-GDP

Ran in nucleus

• Ran guanine nucleotide exchange factor (Ran-GEF) puts new GTP onto Ran-GDP

• produces Ran-GTP

nuclear import process

1. the cargo (with NLS) binds to the the nuclear import receptor

2. receptor carries the cargo across FG filaments of the NPC

3. once in nucleus, Ran-GTP binds to the receptor

   1. receptor releases cargo

4. import receptor + Ran-GTP get shuttled back to the cytoplasm through the NPC

5. once in cytoplasm, Ran-GAP hydrolyzes Ran-GTP to Ran-GDP

   1. Ran-GDP dissociates from receptor

6. receptor free to bind to another cargo

nuclear export process

1. Ran-GTP binds to the nuclear export receptor in the nucleus

2. Ran-GTP-export-receptor binds to the cargo (with NES)

3. receptor carries cargo + Ran-GTP across the FG filaments of NPC

4. once in cytoplasm, Ran-GAP hydrolyzes Ran-GTP to Ran-GDP

   1. receptor releases cargo + Ran-GDP

5. receptor gets shuttled back into nucleus through the NPC

what type of process is protein translocation into the ER

• co-translational

• polypeptide gets inserted into ER lumen as it is being synthesized by the ribosome

what facilitates translocation

protein translocator

protein translocator

• transmembrane protein in the ER membrane

• has a channel for unfolded proteins to pass through

ER import signal

• patch of hydrophobic aa

• protein translocator holds this as the rest of the polypeptide gets inserted into the ER lumen

protein co-translocation into ER process

1. ribosome beings translating the mRNA and ER signal sequence comes out

2. signal recognition particle (SRP) binds to the signal 

3. SRP carries ribosome to the SRP receptor on the ER membrane 

   1. translation temporarily stops since SRP also blocks EF-Tu and EF-G binding

4. SRP receptor interacts with the protein translocator 

   1. ER signal is passed on to the protein translocator

5. SRP and SRP receptor dissociate from the ribosome

6. translation resumes

   1. peptide is inserted into the ER lumen through the protein translocator

transmembrane proteins with alpha helical anchor

also serves as ER signal sequence

how is a single pass protein generated

protein translocator releases this transmembrane alpha helical anchor/ ER signal into the membrane after translation is complete

how are multi pass proteins generated

if multiple transmembrane alpha helical anchors are translated, they each get released into the membrane 

how does vesicular transport work

• cargo is enclosed in a lipid vesicle that buds off from the original organelle 

• vesicle fuses to the lipid bilayer of the target organelle, releasing the cargo

NLS aa sequence

lys lys lys arg lys