BIO230 L9 Protein Sorting

post translational process of protein sorting

proteins fully synthesized in the cytosol before sorting

mitochondria, plasmids, nucleus, peroxisomes all fall under this category

co-translational process of protein sorting

endoplasmic reticulum falls under this category — proteins are associated with the ER during protein synthesis

what type of transport occurs between which organelles? (colour coded flowchart)

gated transport

proteins moving between cytosol and nucleus through the nuclear pore complex

nuclear pore complex

selective transport of macromolecules

free diffusion of molecules <5000 daltons

transport of cargo occurs in both directions

made up of nucleoporins that facilitate transport with the nuclear import/export receptor

nuclear import receptor

a protein in the cytosol which binds to the nuclear localization signal in cargo (or with help from adaptor proteins) 

once bound, binds to nucleoporins in the nuclear pore complex and transports into the nucleus

nuclear import of cargo proteins

1. Nuclear import receptor

   • binds cargo in cytosol

2. Receptor + cargo move to nucleus

3. Ran-GTP binding causes cargo release

4. Empty import receptor + Ran-GTP

   • move to cytosol

5. Ran Binding Protein and Ran-GAP

   1. promote:

      • GTP hydrolysis

      • release of import receptor

nuclear export of cargo proteins

1. Nuclear export receptor

   • binds Ran-GTP + cargo in nucleus

2. Receptor + cargo + Ran-GTP move to cytosol

3. Ran Binding Protein and Ran-GAP promote:

   • GTP hydrolysis

   • release of cargo

   • release of export receptor

4. Empty export receptor returns to nucleus

nuclear export receptor

a protein in the nucleus which binds to the nuclear export signal in cargo

once bound, binds to nucleoporins in the nuclear pore complex and transports into cytosol

newly assembled ribosomal subunits, RNA, or proteins with regulated nuclear import & export may be transported

nuclear localization signal

an amino acid sequence in a protein that binds to the nuclear import receptor

rich in Lys and Arg

Ran GTPase

an enzyme required for nuclear import and export

(GTP) causes cargo release by the NIR in the nucleus and (GDP) dissociates in cytosol

causes cargo binding to the NER in the nucleus and dissociates in cytosol

regulated by Ran-GAP (in nucleus) and Ran-GEF (in cytosol)

Ran-GAP (GTPase-Activating Protein)

an enzyme residing in the cytosol

promotes GTP hydrolysis by Ran and release of cargo and export receptor in nuclear export, import receptor in nuclear import

Ran-GEF (Guanine Nucleotide Exchange Factor)

an enzyme residing in the nucleus

promotes exchange of GDP for GTP by Ran

NTF2 (Nuclear Transport Factor 2)

transports Ran-GDP from the cytosol back into the nucleus

effectively creates directionality/a concentration gradient for Ran GTP/GDP

NFAT import/export cycle

high [intracellular Ca2+] → nuclear import

low [intracellular Ca2+] → nuclear export

transmembrane transport

requires protein translocators — transports generally unfolded proteins across the membrane

occurs in ER, mitochondria, plastids, peroxisomes

why mitochondria and chloroplasts are special cases of transmembrane transport

mostly have their own genome and ribosomes but most proteins are nuclear encoded

have many membranes to pass through

importing proteins into the mitochondrial matrix

TOM and TIM23 complexes

protein remains unfolded in cytosol with use of cytosolic hsp70 chaperones

mitochondrial signal sequence is an N-terminal amphipathic alpha helix that binds receptor and is cleaved upon entry of the matrix

proteins can be further sorted

TOM complex

protein translocator in the mitochondria

on the outer membrane of the mitochondria

TIM23 complex

protein translocator in the mitochondria

on the inner membrane of the mitochondria

importing proteins to the chloroplast

TOC and TIC complexes

chloroplast signal sequence is an N-terminal amphipathic alpha helix that binds receptor in the TOC complex and is cleaved upon entry of the chloroplast

thylakoid signal sequence is a hydrophobic signal sequence unmasked when first signal is cleaved

TOC complex

protein translocator in the chloroplast

has a separate receptor protein that binds precursor

on the outer membrane of the chloroplast

TIC complex

protein translocator in the chloroplast

on the inner membrane of the chloroplast

uses GTP or ATP dependent translocation into the stroma

importing proteins into the thylakoid

hydrophobic thylakoid signal sequence is unmasked when chloroplast signal sequence is cleaved

three different routes to translocate protein into thylakoid space

sorting proteins to the peroxisome

peroxisomal targeting signal contains 3 amino acids at C terminus (SKL)

protein is folded

transported across membrane by a large translocator complex

vesicular transport

vesicles move protein between compartments