2. Protein Targeting - Endoplasmic Reticulum

secretion to the ___ or ___ is the default situation for lipids and proteins synthesized in the ER

plasma membrane or outside world

proteins destined for the cytoplasm have no ___

targeting sequences- this is the default pathway

larger proteins destined for the nucleus have an ___

NLS and are taken up fully folded by nuclear pores (gated transport)

proteins targeted for the ER also have ___

signal sequences and are translocated across the ER membrane into a topologically distinct compartment (transmembrane transport)

ER functions (4)

1. site of protein synthesis for the endomembrane system

2. site of lipid synthesis

3. Ca++ storage

4. enzyme storage in some cell types (detoxifying enzymes in liver cells)

ER functions: site of protein synthesis for the endomembrane system

• the secretory and endocytic pathways

• ER, Golgi, lysosomes, endosomes, secretory vesicles

• transmembrane and GPI-linked proteins

• soluble proteins in the lumen of the endomembrane system

the ER is an ___, ___ structure

extensive, dynamic

the ER is a network (reticulum) of membrane tubules and sheets that stretches ___

from the nucleus to the outer periphery of the cell

the ER is constantly ___

changing shape, extending and retracting

during cell division, the entire ER network ___

breaks down into vesicles that partition into daughter cells

how many types of ER membranes can be seen in the electron microscope

2 types!! rough and smooth

appearance of rough ER under EM

studded with ribosomes, tends to be near the nucleus but is still extensive

appearance of smooth ER under EM

does not have ribosomes and tends to be in the more peripheral regions of the cell

RER ribosomes are bound to the membrane because of ___

nascent chains (new proteins) they are making

• the proteins are being cotranslationally inserted into the membrane

what does a ribosome do again

translate mRNAs to make proteins

the two types of ER membranes can be separated by ___

gradient centrifugation

gradient centrifugation of the ER membranes

• break cell mechanically

• microsomes form; vesicles containing the membrane components that the ER had

• Rough microsomes - dense due to ribosomes

• Smooth microsomes - lighter due to lack of ribosomes (duh)

what is a polyribosome

A polyribosome, or polysome, is a complex of multiple ribosomes translating a single mRNA strand simultaneously

Free and Bound Polyribosomes

• protein translation is often ___

• mRNAs are often ___

• once a ribosome has moved away from the start point on the mRNA, ___

• many ribosomes can attach to a single mRNA in this way - it is now a ___

• if an ER targeting signal is present in the protein, the ribosome and ___

• free and bound polysomes appear identical except for ___

• RER is efficient because the mRNA tends to remain near the ___

• slow

• long

• another ribosome can attach

• polyribosome or polysome

  • most cellular proteins are made on polysome

• nascent chain dock to the membrane - Bound polysome

• localization and the peptide being made

• the membrane where there are abundant ribosomes being recycled

A signal sequence directs proteins to the ER

• the ER signal sequence: __

  • N-terminal signal sequence for ___ membrane proteins

  • internal sequences for ___ membrane proteins

• signal sequence is __ for ER membrane targeting

• leads to ___ across ER membrane

• N-terminal start transfer sequences are usually ___

• the protein folds in the ___, with the help of ___

• protein is now either ___ or ___

• a stretch of hydrophobic amino acids

  • soluble and some membrane proteins

  • many membrane proteins

• necessary and sufficient

• co-translational translocation

• cleaved by signal peptidase, so the sequence is not found in the mature protein

• lumen, chaperones and other molecules

• soluble in the lumen or associated with the ER membrane

proteins destined for the ER are transported by a ___ mechanism

cotranslational transmembrane

how is the ER targeting signal recognized?

• signal sequence emerges from ribosome

• associates with the Signal Recognition Particle (SRP)

the ___ recognizes the signal sequence

signal recognition particle (SRP)

SRP recognizes the ___

• SRP is found ___ (where?)

• discovered as a factor that allowed ___

• SRP is a ___ particle; RNP = ___ + ___

• binds to both the ___ and ___

• binding of the SRP to the nascent peptide chain halts ___ when the peptide chain is ___ long

signal sequence

• free in the cytoplasm

• attachment of pure ribosomes to ER membranes

• ribonucleoproteinh; RNA + 6 proteins

• signal sequence and ribosome

• translation; 70-100 amino acids long (long enough to protrude from the ribosome)

cotranslational translocation of proteins into the ER (no shot youre remembering this)

• signal sequence emerges from ribosomes

• associates with the SRP

• SRP binds to signal sequence and ribosome

  • causes translation to pause

• SRP associates with SRP-receptor in membrane and the ribosome docks onto translocon channel

  • this tight dock is what protects proteins from protease in our assays

• translocon pore through the membrane opens

  • normally closed otherwise small particles would leak out and cytoplasm/ER compartments would be in equilibrium

• signal sequence and adjacent nascent chains inserted pore

• both SRP and SRP-receptor release from ribosome and translocon

• translation resumes through the membrane pore and protein enterss lumen

• SRP/SRP-receptor binding, ribosome docking, and SRP/SRP receptor release is somehow regulated by GTP binding/hydrolysis by BOTH SRP and SRP-receptor

• N-terminal sequence is cleaved by signal peptidase

• protein synthesis is completed with ribosome still docked

• translocation channel closes and ribosome unlocks

how do you demonstrate co-translational transport?

use microsomes and protease protection experiments to ask whether proteins are ever exposed

demonstrating co-translational transport using microsomes and protease protection experiments

• you can mix mRNA encoding an ER-targeted protein with ____ and synthesize a protein (__)

• if you added a protease to the tube during or after the synthesis, you find that your protein ___

• if you repeat the experiment using microsomes, you find that your protein ___

• if you add detergent during or after synthesis, ___

• Conclusion:

  • the proteins are ___ during their synthesis - transport into the lumen of the membranes occurs ___ synthesis, aka: ___

• ribosomes, ATP, tRNAs, and AAs (in vitro)

• would be degraded (run a gel to determine size)

• is not degraded, even if the protease is present during synthesis

• in the presence of protease, the proteins are of course degraded

• Conclusion:

  • protected; during; cotranslation translocation

a) mRNA + ribo + (ATP, tRNA, AAs etc) =

b) mRNA + ribo + (etc) + SRP =

c) mRNA + ribo + (etc) + SRP + SRP receptor =

d) mRNA + ribo + (etc) + sMicro + SRP + SRP receptor =

e) mRNA + ribo + (etc) + sMicro =

Full Protein, Protease Sensitive

a) +, +

b) 70-100 AAs, -*

c) +, +

d) +**, -

e) +,+

*70-100 amino acids protected by SRP and the ribosome itself

**slightly shorter due to cleaving og signal sequence by peptidase in sMicro membrane

Recap: sequential reactions and cotranslational translocation into the ER

• Translation begins, exposing ___

• Free SRP in cytoplasm binds ___

• As a results of binding, SRP causes ___

• SRP now binds ___ on the cytoplasmic side of the ER membrane, ribosome docks onto ___

• __ and __ inserted into translocon

• SRP/SRP-receptor dissociate and ___, ready to be used again

• as a result of SRP release, translation ___; with signal peptide bound in translocon, ___ translocates alongside

• _____

• signal peptide

• signal and ribosome

• translation to stop

• SRP-receptor; translocon

• are released from ribosome

• now resumes; polypeptide chain

• N-terminal signal peptide cleaved

the N-terminal signal sequence is cleaved off by ___

signal peptidase

what is signal peptidase

a protease that recognizes a specific sequence and is embedded in the ER membrane and associated with the translocon complex

the cleaving of the N-terminal signal sequences causes what

• releases soluble proteins into the lumen of the ER

• proteins made in this way can eventually be secreted by the cell, or if they have appropriate additional signals, reside in the ER, Golgi, lysosomes, etc

if the signal sequences was not removed, what happens

the protein would stay stuck in the membrane, held by the signal sequence

what are the different types of transmembrane proteins

Type I: single pass; N-terminal inside cell, C-terminal outside (signal sequence cleaved)

Type II: single pass; C-terminal inside cell, N-terminal outside

Type III: single pass; N-terminal inside cell, C-terminal outside

Type IV: multipass; N-terminal inside cell, C-terminal outside

GPI-linked proteins

one way to make single pass transmembrane proteins: ___ (Type I)

encode a stope transfer sequence

describe a stop transfer sequence

hydrophobic stretch of amino acids that is both a signal and the transmembrane domain

Stop-transfer Sequence and Type I Single Pass Protein

• N-terminal sequence directs peptide to membrane

• transfer begins

• stop transfer sequence is made; ___ stops

• hydrophobic stop transfer sequence moves laterally out of translocon and ___

• protein continues to be made ___ (where?)

• ___/___ is removed

• finished protein has N-terminal domain in ___ and C-terminal domain in ___

• transfer to lumen

• becomes anchored in the membrane

• on the outside; “squeezes out” between ribosome and translocon seal

• N-terminal signal/start transfer

• in ER lumen; in cytoplasm

what would happen if you removed the stop transfer sequence from a Type I transmembrane protein?

it would go into the lumen and act more like a secreted protein

what would happen if you added a stop transfer sequence to a soluble ER protien?

it would act like a Type I transmembrane protein

another way to make a single pass transmembrane protein (Type 2 & 3): ___

the internal start transfer sequence

Start-transfer Sequence and Type 2 & 3 Single Pass Protein

• no ___

• an internal start transfer sequence aka. signal anchor sequence

• sequence is both the ___ and ___

• association with the ___ is delayed as the signal is further away from the N-terminus of the nascent chain

• sometimes the___ ends up inside the lumen and sometimes in the cytoplasm; topology depends on ___

• these signals are not ____, they are ___

• N-terminal signal sequence

• signal and the transmembrane anchor

• SRP and ER membrane

• N-terminus; the orientation of a stretch of positively charged amino acids near the TM domain.

  • these + amino acids will be positioned on the cytoplasmic side of the membrane by the translocon

• cleaved off, TM domains

Type II transmembrane protein has ___

positively charged amino acids residues at the N-terminal end of its signal sequence

Type III transmembrane protein has ___

positively charged amino acid residues at the C-terminal end of its signal anchor sequence

Hydrophobic alpha helices as signals for protein transfer

• N-terminal signal sequences, internal start transfer (signal anchor) sequences, and interna. stop sequences are all ___

  • N-term signal sequence will ___

  • internal signals are the ___

• recognized by SRP, allowing ___

• transmembrane domains are ___ while the protein ___

  • for multipass membrane protein topology (Type IV i think), the key is ___

• hydrophobic (usually alpha helix)

  • be cleaved, others are not

  • transmembrane domains

• ribosome docking and arrangement of signals in translocon

• released laterally from the translocon into the ER membrane; continues to be made, either into the lumen or on the cytoplasmic side

• order and orientation of these topogenic sequences

for multipass transmembrane protein transport (Type IV i think), the key is ___

the order and orientation of these topogenic sequences

in some eukaryotes such as yeast, some proteins are ___

translocated post-translationally

in post-translational translocation, how is the polypeptide chain “pulled” through the translocon and into the ER lumen

ADP clamps down on lumen side, random motion of the chain only allows it go inward

“thermal ratchet mechanism”

Glycosylation of Proteins

• most proteins imported into the ER become ___

  • critical for quality control of ___ in the ER

  • further processing of these sugars in Golgi for ___

• Oligosaccharyltransferase is an ___ associated with the ___

• the sugars are added ___

• N-linked Glycosylation; the signal for glycosylation is ___

• the oligosaccharide is ___ in the ER and added ___

• multiple oligosaccharides are often ___

• this only happens in the ___

• glycosylated

  • folding

  • proper protein function

• integral membrane protein; translocon complex

• cotranslationally during import

• the amino acid sequence Asn-X-Ser and addition is to a nitrogen in Asn

• preformed; as a block

• added to a protein, each Asn-X-Ser will get its own oligosaccharide

• lumen of the ER; there are no oligosaccharides of this sort on the cytoplasmic proteins

most proteins imported into the ER become ___

glycosylated

what is oligosaccharyltransferase

integral membrane protein associated with the translocon complex

a glycoprotein to look at :)

Source of Oligosaccharides

• the oligosacc is built initially ___

• partway through construction it is moved ___

• construction is finished ___

• the entire oligosacc is then transferred ___

• in the cytoplasm on a lipid carrier (dolichol)

• across the membrane by transport-like proteins to the ER lumen

• on the ER lumen side

• to the protein during translation (co-translocationally) by oligosaccharyltransferase

cotranslational glycosylation happens via ___

oligosaccharyltransferase

how does ADP “pull” a polypeptide chain through the translocon in post-translational translocation

thermal ratchet mechanism

GPI

glycosyl-phosphatidyl-inositol

some proteins are held in the membrane by ___

lipid anchors

Lipid anchored proteins

• GPI anchoring is done in the ___

• begins as TM protein; an enzyme cleaves the ___ and attaches it to the ___ at the same time

• GPI-anchored membrane proteins face the ___ or ultimately the ___ if in plasma membrane

• these proteins can be rapidly released from membranes by ___

• ER

• the protein; GPI anchor

• lumen; extracellular space

• cleaving the anchor

Protein folding in the ER

• __ in the ER lumen aid in folding

  • __ - the family of proteins that either aid in protein folding or protect the cell from unfolding in response to heat stress (heat, chemical)

  • __ are constitutively expressed and bind unfolded or misfolded proteins in this compartment

• Chaperone proteins

  • Heat shock proteins (Hsc, Hsp)

  • Similar ER localized chaperones such as BiP (Hsp70 family member)

what in the ER lumen helps aid in protein folding

chaperone proteins

Protein folding in the ER

• Protein disulfide isomerase (PDI)

  • the lumens of ___ are harsher, more oxidizing environments that the cytoplasm

  • __ in oxidizing conditions can help to stabilize protein structure

  • proteins have evolved differently depending on where ___

  • PDI catalyzes ___

  • the “shuffling” of disulfide bonds by PDI gives proteins ___

• endomembrane compartments, and the extracellular world

• Disulfide bonds S-S that form between cysteines

• they are going to fold!

• a chance to be properly folded

PDI

protein disulfide isomerase

proteins that fold incorrectly are recognized as ___

“WRONG”

ERAD

ER-associated protein degradation

(think of “eradicate”)

what does it mean for something to be polyubiquitinated

monomers of the small protein molecule ubiquitin are added

ER-associated protein degradation (ERAD)

• misfolded proteins are ___, ___, and ___ back across the ER membrane to the cytoplasm

• during ___ the proteins become polyubiquitinated; ___

• the cytoplasmic proteasome recognizes ___ and ___

• recognized, modified, “dislocated”

• dislocation; monomers of the small protein molecule ubiquitous are added in a chain by an E3-ubiquitin ligase

• polyubiquitin; degrades the protein

the calnexin/calcreticulin cycle of quality control in the ER

• ERp57, a ___

• Calnexin, a ___

• Glucosidase II, an ___

• UGGT, a ____

• a PDI-type protein

• a chaperon protein that can bind oligosaccharides (a lectin)

• enzyme that recognizes if protein is folded correctly

  • if not: glucose reattached

  • if so: export to golgi

the accumulation of misfolded proteins in the ER triggers ____, including increased transcription of genes encoding ___

an unfolded protein response; chaperones and dislocation machinery