MBB231 - Lecture 10: Endomembranes - ER import

what are the two locations within the cell where polypeptides are synthesized

• rough endoplasmic reticulum

• free roaming ribosomes in the cytosol

what kind of proteins are synthesized in the rough endoplasmic reticulum

• secreted proteins (proteins that are excreted out of the cell to be used elsewhere in the body)

• transmembrane proteins (integral membrane proteins)

• soluble proteins

what kind of proteins are synthesized on cytosolic ribosomes

• proteins that stay in the cytosol

• peripheral proteins (only stays on cytosolic side of the membrane)

• proteins that travel → nucleus

• proteins that are incorporated into peroxisome, chloroplasts, and mitochondria

what are the two pathways involved in protein sorting

• co-translational import

• post-translational import

what happens in the co-translational pathway

• proteins carrying an ER signal sequence directs the ribosomal polypeptide complex → rough ER

• translation is completed at the rough ER

• in the ER - proteins are further processed + sorted in the ER and golgi

what occurs in the post-translational import

• proteins lacking an ER signal sequence - complete their synthesis on ribosomes that free roam the cytoplasm

• proteins are released into the cytoplasm

• proteins with an organelle specific sorting signal → imported into the specific organelle

why do cytoplasmic proteins do not have a sorting signal?

they do not need a sorting signal as the cytosol is the default location → are left in the cytosol where they are made (by ribosomes)

what are the components of the endomembrane system

• Rough / smooth ER

• golgi complex

• endosomes

• lysosomes

what are rough / smooth ER and the golgi complex sites used for

used for protein (+lipid) synthesis. processing and sorting

what do endosomes do

carry and sort material brought into the cell

what do transition/ transport/ secretory vesicles do

move molecules between compartments and the plasma membrane

where does the endomembrane system start

starts at the endoplasmic reticulum

what does the ER consist of

tubular membranes and flattened sacs → cisternae

what is the internal space of the ER called

lumen

the co-translational pathway is utilized by what organelle

by ribosomes synthesizing polypeptides that will be exported from the cell/intracellular compartments (i.e. lysosomes/endosomes/golgi)

what is the signal hypothesis

explains how a cell routes newly manufactured proteins to their correct destination using a built in “address tag” → made up of amino acids

what happens if a sorting signal is deleted

the protein is not recognized by transport machinery and typically remains in the cytosol

what happens if the sorting signal is transplanted to another protein

the recipient protein of the sorting signal is recognized by transport machinery → protein is then directed to the sorting signal’s final destination

what is co-translational translocation of soluble proteins - what is the point

ER signal sequence directs the complex (contains: mRNA, polypeptide, ribosome) to the surface of the rough ER

in the complex: where does the mRNA come from

is produced by the nucleus and released to be translated by the ribosome to form new proteins

explain the process of co-translational translocation of soluble proteins

signal sequence is recognized by the SRP (signal recognition particle):

• polypeptide synthesis continues until the ER signal sequence is formed

• SRP binds to the signal sequence → blocks / pauses translation

• SRP binds to the ribosome

→ directs the entire complex to the translocon in the ER membrane

at the translocon:

• complex binds to the SRP receptor on the cytoplasmic surface of the ER membrane (close to the translocon)

• SRP is released - ER signal sequence is inserted into the translocon

in the translocon:

• contact of the ER signal sequence with the inside of the translocon → displaces the plug

• conformational change occurs → results in the channel to open to the lumen of the ER

• polypeptide resumes synthesis → passing into the lumen of the ER

during synthesis:

• signal peptidase cleaves the ER signal sequence → sequence quickly degrades

• chaperones - bind to the newly synthesized polypeptide and facilitate proper folding and to prevent aggregation

after protein synthesis:

• once synthesis is complete - polypeptide is released into the lumen

• ribosome detaches from the ER membrane → releases the mRNA

what do G-proteins act as

molecular switches → switches between two conformations (GTP- / GDP- bound state)

what processes are G-proteins typically used for

• cell signaling

• cell division

• protein synthesis

• cargo selection

• vesicle fusion

how is SRP / SRP receptor an example of G - proteins

in GTP bound state → they have high affinity for each other

• hydrolysis of the GTP state causes a change in conformation + state → reduces the affinity for each other resulting in them releasing

what are transmembrane proteins also known as

integral membrane proteins

describe the orientation of the hydrophobic alpha - helical in transmembrane proteins

• hydrophobic segments point outwards from the helix → to complement the hydrophobic fatty acid tails in the center of the membrane

• backbone of the helical are naturally hydrophilic → twisting allows all the hydrophilic components to be protected through hydrogen bonding

what are the two types of transmembrane proteins - what is the difference

single pass: protein chain cross the cell membrane only once

• usually has one hydrophobic alpha helix region in the membrane

multi pass: protein chain zags back and forth → crossing the cell membrane two or more times

• contains the multiple hydrophobic alpha helices (or beta barrels) that weave through the membrane

what is the difference between type 1 and type 2 single pass transmembrane proteins

type 1:

• N terminus location: outside of the membrane (ER lumen)

• C terminus location: inside of the membrane (cytosol)

• has an N-terminal signal that is cleaved later on

• has a hydrophobic stop transfer sequence - helps it anchor to the membrane

type 2:

• N terminus location: inside the membrane

• C terminus location: outside of the membrane

• has a single hydrophobic region near the N-terminus → acts as the membrane anchor + start signal

what happens if you have one signal (start only) or two signals (start and stop)

1 signal (start only) - protein goes all the way through into the ER of the lumen

2 signal (start + stop) - protein gets stuck halfway through

• released laterally - hydrophobic components dissolve with the fatty acid tails → results in a membrane bound protein

how is orientation decided for single pass transmembrane proteins

• outer face of the ER membrane + translocon have a negative charge due to phosphatidylserine

• positive charged amino acids flanking the hydrophobic anchor are attracted to the negative charge → they stay on the cytosol side of the membrane

how is multi pass transmembrane protein orientated

• orientation is determined by the charge / orientation of the first transmembrane domain

• after the first domain → each domain is orientated in the opposite way from the previous domain

What is N-linked glycosylation

attachment of an oligosaccharide directly to the nitrogen of the amino acid asparagine

where does N-linked glycosylation occur in

occurs in the inside of the lumen of the endoplasmic reticulum

what is co-translational glycosylation

when the oligosaccharide is added to the recipient protein as it is being synthesized

what could be the possible reason for the initial processing in the ER of an N-linked oligosaccharide

3 glucose residues + 1 mannose residue is removed - possible the sugars are used as a signal indicating a mature oligosaccharide is formed

what is the purpose of the re-addition of a glucose residue in the processing of N-linked oligosaccharides

the glucose residue plays a role in protein folding

how is glycosylation modified in the golgi

O-linked sugars are added to the serine’s + threonine’s hydroxyl groups

what is the function of the carbohydrate groups (sugars) added in glycosylation + modification of glycosylation

• macromolecule binding site

• help in protein folding

• increases stability

how does the cell ensure newly made proteins entering the ER fold into the right shape

entering the ER: new unfolded polypeptide (protein) chain enters the lumen of the ER with a special sugar tag (from N-linked glycosylation) + one terminal glucose resiude

helper proteins: calnexin & calreticulin - act like chaperones by recognizing and grabbing onto a specific sugar tag on the protein and prevent aggregation from occurring while it folds

PDI - catalyzes the formation of disulfide bonds in the polypeptide → covalent linkages are used between different parts of the protein to lock its 3D shape in place

release + exit: once the protein is folded correctly - enzyme cleaves the glucose residue

• protein dissociates from the chaperones

• leaves the ER

what is BiP (binding protein) and what does it do

it is a chaperone that is abundantly found in the ER lumen

• binds to hydrophobic regions of a polypeptide chain and prevents aggregation of similar regions within the polypeptide region

• prevents interaction between hydrophobic regions of different proteins

• if protein folds incorrectly - interacts with BiP until proper folding is done

how does disulfide bond formation occur with PDI (protein disulfide isomerase)

• disulfide bond in the active site of PDI → transferred to the protein

• PDI goes from oxidized → reduced (re-oxidized by Ero1)

in Eukaryotic cells - where are disulfide bonds formed

only in the ER lumen

disulfide bonds form between which amino acids

between adjacent cysteine groups

how are proteins attached to the GPI anchors in the ER

starting position: newly made protein is anchored in the ER membrane

cut & paste: transamidase (enzyme) simultaneously cleaves the precursor protein and transfers the new C - terminus to the amino (NH2) group of the preformed GPI anchor

→ upon being cleaved the part of the precursor protein in the membrane is left to degrade

what is the unfolded protein response

a stress response pathway in which sensor molecules in the ER detect a misfolded protein

• phosphorylate translation factors inhibit further protein synthesis → decrease flow of proteins entering the ER

• upregulate the expression of:

  • ER-based chaperones → help facilitate proper folding

  • transport → moves proteins out of the ER

  • protein degradation machinery

what is ER-associated degradation (ERAD)

component of the unfolded protein response that recognizes misfolded or unassembled proteins

→ misfolded proteins are exported to the cytosol - degraded by proteasomes

where are the majority of lipids made - what are the exceptions that make their own lipids

in the endoplasmic reticulum - exceptions:

• mitochondria

• chloroplasts

• peroxisomes

how do membranes of different organelles have distinct lipid compositions

• selective transport - vesicles can carry specific lipids to other organelles

• local modification - contain certain enzymes that can modify pre-existing lipids into new types

• lipid transfer proteins - special lipid transfer proteins carry individual lipids directly from the ER to other organelles