Protein trafficking (nucleus/cytosol/mitochondria)

Gated transport

cytosol → nucleus

Transmembrane transport

cytosol → ER

cytosol → mitochondria

Vesicular transport

ER → golgi

golgi → cell exterior

Where will a protein w/ a sequence that codes for an amphipathic helix @ the N-terminal reside?

mitochondria

Where will a protein w/ 2 clusters of + charged aa near the N-terminal reside? Does it change locations if the sequence was in the middle?

in the nucleus; no

Where will a protein w/ K-D-E-L @ the C-terminal reside?

should return to the ER

*COP I

Why will NLSs not be cleaved upon import into the nucleus?

the protein wouldn’t be able to return to the nucleus after the nuclear envelope degrades (prophase); it would float freely in the cell before the nuclear envelope reforms (telophase)

Where are resident nuclear and mitochondrial proteins transcribed?

in the nucleus

Where are resident nuclear and mitochondrial proteins translated?

in the cytosol

Structure (nucleus)

• nuclear envelope (inner/outer membrane) - encloses DNA

  • NPCs - penetrate membranes and contain nuclear porins (channels)

Function (nucleus)

stores genetic info, regulates transcription, RNA processing, DNA replication

What molecules are imported into the nucleus?

histones, DNA/RNA polymerases, transcriptional regulators, RNA processing proteins

What molecules are exported into the nucleus?

RNAs, ribosomal protein complexes, rRNA, mitochondrial proteins

Nuclear import

Ran-GDP (G-protein)

only cargo or G-protein are bound to the receptor (one at a time)

Nuclear export

Ran-GTP (G-protein)

both cargo and G-protein are bound to the receptor (at the same time)

GAP (location)

cytosol → if in the nucleus, the [GTP] would decrease and less cargo would be exported

GEF (location)

nucleus (*with chromatin) → if in the cytosol, the [GTP] would increase and less cargo would bind to importins and move into the nucleus

Why are the locations of GAPs and GEFs important?

allow correct movement of nucleotides; form concentration gradient

• Ran-GEF enriched in Ran-GTP

• Ran-GAP enriched in Ran-GDP

What happens if GEF is dysfunctional?

import would happen @ a slower rate since it would take longer for GDP to dissociate and GTP to rush in; export would most likely stop b/c cargo proteins would remain bound in the nucleus

Importin/cargo

higher concentration in the cytoplasm (passive)

Ran-GTP/importin

higher concentration in the nucleus (passive)

Ran-GDP

higher in the cytoplasm (passive)

mRNA export

not use Ran-GTP/GDP; NXF/T proteins bind to mRNA and Dbp5 provides energy (ATP) for proteins to dissociate from mRNA

Phosphorylation mechanism (nuclear transport)

1) NFAT phosphorylated in cytosol

2) calcineurin (phosphatase) removes phosphate and binds/blocks the export signal

3) conformation change of NFAT exposes nuclear import signal → travels through (gene transcription)

4) low Ca2+ removes calcineurin and ATP/protein kinase rushes in

5) addition of phosphate shifts conformation and exposes export signal (starts over)

Mitochondria (structure)

inner membrane - boundary membrane, cristae, crista junction

outer membrane

intermembrane space

matrix

Mitochondria (function)

powerhouse of the cell (ATP prod.), makes phospholipids

TOM

initiates transport across outer membrane

TIM 22

facilitates inner membrane insertion of multi-pass transmembrane proteins (nucleotide antiporters)

TIM 23

assists w/ transport of proteins into matrix and some inner membrane transmembrane proteins

OXA

initiates insertion of transmembrane proteins coded for by mitochondrial DNA

SAM

facilitates insertion of beta-barrel pores (outer membrane)

How are TOMs and TIMs related?

move proteins to the matrix (intermembrane space)

How does a mito transport protein know it’s importing a transmembrane protein?

it recognizes the target sequences (amphipathic, at least 1 hydrophobic stop transfer)

*allows it to stay embedded

How many hydrophobic sequences are in soluble proteins in the mito matrix?

none; should have been cleaved off by the time it reaches the matrix (float freely)

Mitochondrial transport proteins

TOM, TIM22, TIM23, OXA, SAM

Where will a protein coded by nuclear DNA w/ a mutated OXA sequence (no amphipathic helix) end up?

it will remain in the cytosol (mito proteins fully translated in the cytosol)

Post-translational process

sorting proteins to the mitochondria

Unstructured domains

allow small molecules to diffuse through NPC, but larger proteins enter via importin or other nuclear chaperones