Lecture 15 Intracellular Trafficking to ER

Majority of proteins are synthesized by

(slide 6)

free cytosolic ribosomes

-cytosolic proteins

cytosolic peripheral membrane proteins

-nuclear proteins

-proteins targeted to mitochondria, chloroplasts, and peroxisomes

(slide 7)

free ribosomes

One-third of all proteins are synthesized by ribosomes on

(slide 6)

the ER membrane

• Secreted proteins

• Integral membrane proteins

• Soluble proteins of organelles

(slide 7)

Rough ER

process by which newly synthesized proteins are delivered to their appropriate destination in cells

(slide 9)

protein targeting

present within the polypeptide targets the protein to different locations

(slide 10)

signal sequence

ER signal sequence -------> ER

(slide 10)

Mitochondrial signal sequence

(slide 10)

Mitochondria

Nuclear signal sequence

(slide 10)

Nucleus

No signal sequence

(slide 10)

Cytoplasm

inserted into the lipid bilayer

(slide 10)

Membrane proteins

the entire protein is translocated across the membrane into the aqueous interior of the organelle

(slide 10)

Water-soluble proteins

targeted to the endoplasmic reticulum, mitochondria, chloroplasts, peroxisomes, and nucleus through signal sequences

(slide 11)

Proteins

NH3+ end

6-12 hydrophobic aa,1 basic aa

chain of 6–12 Hydrophobic amino acids — such as leucine (L), isoleucine (I), valine (V), phenylalanine (F), or methionine (M). 1 Basic amino acid (aa) — often lysine (K) or arginine (R)

(slide 11)

ER

NH3+ end

3-5 non-consecutive Arg, Lys

(slide 11)

Mitochondria

NH3+ end
rich in Ser, Thr

(slide 11)

Chloroplast

COO- end

Ser-Lys-Leu

(slide 11)

Peroxisome

Internal

5 basic aa/2 small clusters of basic aa

(basic (positively charged) amino acids — mainly lysine (K) and arginine (R). single stretch of about five positively charged residues. two short clusters of positively charged residues separated by a few other amino acids)

(slide 11)

Nucleus

Vesicle-based trafficking of Proteins (Secretory Pathway) involves transport of proteins from the ER to their destination with the help of

(slide 15)

membrane-bound vesicles

Secretory pathway begins in

(slide 15)

ER

A network of membranous tubules within the cytoplasm of a
eukaryotic cell

Acts as an intracellular transporting system or canal

(slide 17)

Endoplasmic Reticulum

studded with membrane bound ribosomes

(slide 17)

Rough endoplasmic reticulum

Lacks ribosomes.
Makes phospholipids and fatty acids.

(slide 17)

Smooth endoplasmic reticulum

Add media containing radioactive amino acid

(e.g., radioactive leucine) (slide 20)

Pulse

Remove media containing radioactive amino acid
(radioactive leucine)
Add media containing non-radioactive amino acids (slide 20)

Chase

The squiggles show radioactive proteins (slide 23)

3 minutes pulse, 0 min of chase

Majority of radioactive proteins have moved to the periphery of the Golgi complex

(slide 24)

7 minutes Chase

Radioactive proteins are in vesicles moving
towards the plasma membrane

(slide 25)

37 minutes Chase

Radioactive proteins are in the extracellular
space

(slide 26)

117 minutes Chase

synthesized on ribosomes bound to the cytosolic face of the ER membrane

(slide 27)

Secretory proteins

The polypeptides produced by ribosomes end up within the lumen of the

(slide 27)

endoplasmic reticulum

What experiment showed secretory proteins are translocated from the cytosolic side to the lumen of ER

(slide 29)

In vitro reconstitution

translocated into the ER lumen (slide 31)

Soluble (secretory) proteins

embedded in the ER membrane bilayer (slide 31)

Transmembrane proteins

located at the N-terminus of the protein (slide 32)

ER targeting signal sequence

Continuous stretch of 6 – 12 hydrophobic amino acids at the N-terminus

(slide 32)

ER signal sequence

critical for interacting with the machinery that
targets the protein to the ER membrane (slide 32)

hydrophobic core

Is the Signal sequence part of the mature protein

No

Are Secretory proteins incorporated into the lumen of the microsomes (slide 34)

No

The transport of most secretory proteins into the ER lumen begins while the recent protein is still bound to the ribosome (slide 35)

co-translational translocation

Complex of 6 proteins and an RNA molecule (300 bp)

Subunit P54 has a hydrophobic region that interacts with the hydrophobic core of the ER Signal sequence

Other regions interact with the ribosome and the SRP receptor

(slide 37)

Signal Recognition Particle (SRP)

Integral membrane protein on ER membrane
Has two subunits (⍺ and ß)
SRP-polypeptide-ribosome complex bind to ER membrane by docking to the SRP receptor

(slide 37)

SRP Receptor

Channel made up of proteins embedded within the ER

membrane (slide 40)

Translocon

What type of proteins enter the ER lumen through the translocon (slide 40)

Newly synthesized

Translocon is composed of what protein (slide 40)

Sec61

proteins in yeast that enter the ER lumen after translation is completed (slide 47)

Post-translational Translocation

Does Post-translational translocation” use SRP and SRP receptor to bind to Sec61 translocon (slide 48)

No

Post-translational translocation uses what to drive the peptide into the ER lumen through the Sec61 translocon (slide 48)

Sec63 complex and BiP

What has a peptide binding domain and an ATPase domain (slide 48)

BiP

refers to the number of times the polypeptide chain spans the membrane and the orientation of the membrane-spanning segments (slide 53)

Topology of membrane proteins

always faces cytoplasmic (slide 54)

Cytosol

face Exoplasmic side (slide 54)

ER Lumen

the end that faces or is inside the lumen will face (slide 55)

extracellular space

only 1 membrane spanning ⍺-helix (slide 57)

Single Pass

2 or more membrane spanning ⍺-helix (slide 57)

Multi Pass

N-terminal end is inside ER lumen (exoplasmic space) Nexo

C-terminal end is in the cytoplasmic side Ccyto

N-terminal cleavable ER signal sequence

(slide 58)

TYPE I Transmembrane Protein

N-terminal end in cytoplasmic side – Ncyto
C-terminal end in ER lumen (exoplasmic space) – Cexo

(slide 60)

TYPE II Transmembrane Protein

N-terminal end in cytoplasmic side – Ncyto
C-terminal end in ER lumen (exoplasmic space) – Cexo

single “internal” hydrophobic signal-anchor sequence (SA)

(slide 62)

TYPE III Transmembrane Protein

No N-terminal signal sequence
Has a Hydrophobic C-terminus – not available for membrane insertion until protein synthesis is complete and the protein has been released from the ribosome

(slide 65)

Tail-anchored transmembrane proteins

Proteins anchored to the membrane by covalent bonds
with an amphipathic molecule

(slide 69)

glycosylphosphatidylinositol (GPI)

cleaves off the original stop-transfer anchor sequence and transfers the luminal portion of the protein to a preformed GPI anchor in the membrane. (slide 69)

Transamidase