Topic 12: Transport into the ER

Protein targeting map; cytosol___ mitochondria/chloroplasts through

→ ; signal peptide (amphipathic alpha helix)

Protein targeting map; cytosol___nucleus through

$\leftrightarrow$ ; Nuclear Localization Signal (NLS)

Protein targeting map; Cytosol → ____ through ___

Rough ER; signal sequence (hydrophobic alpha helix)

Protein targeting map; RER $\leftrightarrow$ ____; → through ___ and ← through ___

Golgi; default; Soluble/Membrane proteins

Protein targeting map; golgi →

secretory vesicles/plasma membrane/endosomes

Golgi →secretory vesicles through

constitutive secretion

Golgi → endosomes through

M6P protein

Golgi→plasma membrane through

constitutive secretion

Protein targeting map; secretory vesicles →

plasma membrane

Secretory vesicles→ plasma membrane through

Regulated secretion

Protein targeting map; Plasma membrane → ___ through ___

endosomes; endocytosis

Protein targeting map; endosomes$\leftrightarrow$ ___ through ____

lysosomes; endocytosis

When in the map does protein targeting switch to vesicle targeting

Rough ER$\leftrightarrow$ Golgi, everything after that is also vesicle

Protein construction during nuclear transport

Transport occurs after translation, protein is transported fully folded

Protein construction during mitochondrial transport

Transport occurs after translation, protein is unfolded during transport

Protein construction during ER transport

protein is cotranslated (moved as it is made)

Once a protein is in the ER, transport to other organelles occur through

vesicles

microsomes

vesicles derived from ER by biochemical prep

True or false; ribosomes on RER come from same pool of subunits used to translate proteins in the cytosol

true; theyre still ribosomes

What specifies delivery to the ER

Signal sequence/peptide

Evidence for cotranslational ER import

RER microsomes are added DURING translation, so proteins were protected against protease bc they were inside of microsomes (vesicles derived from ER)

When protease was added testing mitochondrial and nuclear import, the protein degraded bc they were not within microsomes

3 points of the signal hypothesis

1. Signal for translocation of a protein into the ER resides in the nascent polypeptide, in the form of a leader “pre” sequence of signal peptide

2. Translocation of the polypeptide across the ER membrane is cotranslational, unlide import into nucleus/mito/chloro

3. Signal peptide is cleaved post-translationally in the ER lumen by signal peptidase (like mito/chloro signals

Signal sequences can be located

at amino end of alpha helix or internal/within the alpha helix

True or false; Internal signal sequences are cleaved by signal peptidase, N terminal signal sequences are not cleaved

FALSE; Internal signal sequences are NOT cleaved, N terminal signal sequences are cleaved by signal peptidase

!Nascent Protein!

Protein that is still/actively being synthesized by the ribosome

Requirements for targeting and translocation into the ER

1. Signal sequence; hydrophobic alpha helix in nascent protein, often near N terminus

2. Signal recognition particle (SRP); cytoplasmic complex of protein and RNA, binds signal sequence

3. SRP receptor; integral ER membrane protein

4. Translocon; Protein translocator- an aqueous channel through ER membrane

Step1 in targeting to RER

Translation exposes signal sequence outside of ribosome

Step 2 targeting to RER

SRP binds the signal seq in nascent protein, temporarily pauses translation

Step 3 targeting to the RER

SRP-arrested ribosome binds SRP receptor in ER membrane (targeting)

Step 4 targeting to RER

Ribosome and polypeptide handed to translocon- SRP is recycled, translation begins and translocation begins

Step 1 of import into soluble proteins

Signal sequence targets nascent protein to ER

Step 2 of import of soluble proteins

Signal sequence cleaved by signal peptidase

step 3 of import of soluble proteins

Translation and translocation are complete, releasing polypeptide into lumen of ER

step 4 of import of soluble proteins

Signal peptide is degraded

Protein folding in the ER is assisted by

BiP

BiP

Binding protein in ER lumen binds nascent polypeptide as it is being translocated, and assists in folding

Release of BiP from folded polypeptide requires

ATP