Part 2: Biogenesis of the Cell Membrane

Microsomes

• small vesicles produced upon breaking the ER by sonication

• used to help explain the molecular aspect of ER transport

Signal Sequences in 1970s

• signal sequences were discovered in the 1970s

• the mRNA encoding a secreted protein was translated by ribosomes in vitro

• w/o microsomes, the protein synthesized was slightly larger than the secreted protein

• with microsomes the protein synthesized was slightly shorter

Blobel/Signal Hypothesis

• he discovered an N-terminal signal that governs protein targeting to the ER and discovered the ER transport 

• signal hypothesis: there is a signal sequence that directs a protein to the ER membrane; when protein is transported, the signal sequence is then cleaved off

Signal Sequence

• a short N-terminal a.a sequence, typically Signal Sequence~20-25 residues

  • placing the N-terminal ER signal on a cytosolic protein redirects the protein to the ER

• the sequence varies in a.a, but each:

  • has 7-15 nonpolar a.a’s at its center 

  • a + charged N-terminal

  • a peptidase cleavage site located 3-7 a.a after the hydrophobic sequence

• Signal Sequencethere is no consensus sequence, the physical properties matter more than the exact a.a sequence

• the signal sequences of all proteins having the same destination are interchageable

Signal Sequence Recognition

• signal sequences are recognized by complementary receptors, called the translocons

• signal peptidases remove the signal sequence during or soon after the transport process is complete

• signal peptidases are located on the trans side of the membrane

Precursor vs Mature Protein

P (precursor): the full length protein with its signal peptide still attached 

M (mature): the processed protein inside the microsome, and the signal peptide is removed by signal peptidase 

Blobel’s Experimental Goal: Recreate ER Transport in vitro

• cells were broken open (homogenized)

• the ER fragments reseal into microsomes (~200nm)

• microsomes are relatively easy to purify by equilibrium sedimentation on sucrose gradient

• microsomes behave and function like mini-ERs:

  • translocation, protein glycosylation, Ca²⁺ uptake and release, lipid synthesis

• the interior of the microsome is equivalent to the internal space of ER (lumen)

The Experiment Didn’t Work

• proteins were synthesized in vitro using ribosomes and radioactive methionine (35S Met), allowing the proteins to show up on film after SDS-PAGE

• then microsomes were added after the proteins were already made

Result: no transport occurred, proteins with signal sequences stayed outside the microsomes

Why? Because ER transport must occur during translation, not after

• the ribosome needs to be attached to the microsome membrane while the protein is being synthesized, allowing the nascent chain to be threaded directly into the lumen

SDS PAGE of the Experiment that didn’t work

Protease: added at the end, digests proteins that are outside the microsome

Detergent: breaks open microsomes, exposing the inside. Now everything can be digested

• SDS-PAGE shows which radiolabeled proteins are protected (inside) vs digested (outside)

• smaller mature (M) band means the protein entered the microsome and had its signal peptide cleaved

  • microsomes present during translation or protease only

• larger precursor (P) bands means the protein stayed outside

  • when microsomes added after translation

SDS PAGE of the Experiment that did work

• when microsomes are added during protein synthesis, the proteins bearing the signal sequence are transported into the microsomes

Microsomes Added during Protein Synthesis Figure

Signal Recognition Particle (SRP): Structure

• an elongated particle made by 6 different polypeptides bound to a small RNA molecule (ribonucleoprotein)

  • the elongated RNA acts like a scaffold to organize SRP proteins

• one end interacts with the ribosome a.a. entry door, the other interacts with the emerging signal sequence

• the signal sequence binding site is a hydrophobic pocket lined by methionine residues

  • Methionine’s flexible side chains provide plasticity, allowing SRP to recognize many different hydrophobic signal sequences.

SRP: Function

• SRP binds to the emerging signal sequence and blocks entry of a.a’s with their tRNA, into the ribosome

  • ∴, SRP blocks the translation of the protein until the ribosome binds to the ER membrane

  • also blocks entry of amino acids with their tRNA into the ribosome

• SRP brings the RNC complex to the ER membrane (spatial effect) by binding the SRP receptor on the membrane

• SRP coordinates protein translocation (temporal effect)

  • pauses translation temporarily after binding the signal sequence, and resumes only after the ribosome binds the SRP receptor and docks to the translocon

SRP directs RNC (ribosome-nascent chain) complex to SRP membrane receptor

SRP: Function FIGURE

Early Model for Co-Translational Translocation

• the ribosome docks on to the ER membrane, and as it makes the protein, it injects the growing chain directly into the ER lumen

• the signal peptide at the start of the protein tells it to go to the ER

• once inside, the signal peptide is cut off by a signal peptidase (enzyme on lumenal side)

• the rest of the protein folds inside the ER lumen

SRP: Continuation of Translation

• once the SRP–RNC complex docks onto the SRP receptor on the ER membrane,  SRP and SRP receptor are dissociated, releasing the ribosome to transfer to the translocon

• translation resumes, and the nascent polypeptide is threaded into the ER lumen thru the translocon channel

• this sequence of events is crucial to prevent protein misfolding/aggregation/activation in the cytosol

• dissociation is regulated by GTP

SecY/Sec61 Translocon

• translocon is also called the Sec61 complex, made up of 3 subunits

  • the polypeptide chain is transferred thru the translocon (a membrane channel)

• prokaryotes: SecY complex (Sec Y, SecE, SecG)

• eukaryotes: Sec61 complex (Sec61⍺, Sec61β, Sec61γ), aka “protein conducting channel (PCC)”

• archaea: SecYβ complex (SecY, Sec61β, SecG)

• provides a pathway through the ER membrane for the growing chain, instead of being released into the cytosol

X-Ray Diffraction

Protein structure can be determined using X-ray diffraction

• X-rays are electromagnetic radiation with a short wavelength

• if a beam of X-rays is directed across a pure protein, most of the X-rays pass thru

• a small fraction is scattered by the atoms in the sample

• if the protein is well-ordered into a crystal, the scattered waves are well-defined

• each spot in the diffraction pattern contains information about the locations of the atoms and can produce a complex 3D electron-density map

Translocon Structure

• the pore is gated by a short helix(green) that keeps the channel closed

• the pore opens when a polypeptide chain traverses the membrane

• the pore ring is made of up 6 Ile’s (hydrophobic residues), that form a gasket around the polypeptide in transit

• the structure allows for translocation and ion tight 

• hourglasss structure 

Translocon: Renoval of the Plug

• removal of the plug is lethal to the cell

• the inner membrane loses its impermeability (molecules equilibrate across membrane) and the cell dies 

Translocon Lateral Gate

• SecY/Sec61α (largest subunit): forms the main pore for polypeptide translocation

• SecE and Secβ/SecG (smaller subunits): stabilize the complex

• Polypeptides pass through the central pore of SecY/Sec61

  • the plug helix blocks the pore when no polypeptide is present, preventing ion leakage.

• lateral gate: SecY/61 channel can open sideways toward the lipid bilayer

  • allows signal sequences to enter the channel and TMS of membrane proteins to exit into the bilayer

Membrane Protein Type 1

• single-pass transmembrane protein with its N-terminus on the extracellular side and its C-terminus on the cytosolic side of the cell membrane

Insertion of a Single Pass Membrane Protein: Details

• the hydrophobic sequence following the signal sequence (7-15 a.a’s) is the stop-transfer sequence

• the stop-transfer sequence remains in the lipid bilayer as a membrane-spanning ⍺ helix (i.e becomes a TMS)

• eg. glycophorin A

Insertion of a Single Pass Membrane Protein: Step 1

• translation begins in the cytosol: ribosome synthesizes nascent polypeptide and the N-terminal signal sequence emerges first

Insertion of a Single Pass Membrane Protein: Step 2

• SRP binds the signal sequence and pauses translation

Insertion of a Single Pass Membrane Protein: Step 3

• SRP-RNC complex docks onto the SRP receptor on the ER membrane

Insertion of a Single Pass Membrane Protein: Step 4

• SRP and SRP receptor dissociate and the ribosome is handed off the Sec61 (translocon), translation resumes

Insertion of a Single Pass Membrane Protein: Step 5

• the N-terminal region is threaded thru the Sec61 channel via hairpin loop into the ER lumen

Insertion of a Single Pass Membrane Protein: Step 6

• signal peptidase in the ER lumen cleaves the N-terminal signal sequence

Insertion of a Single Pass Membrane Protein: Step 7

• the hydrophobic TMS (stop-transfer sequence) of the protein reaches the lateral gate of Sec61, halting further translocation of the polypeptide into the ER lumen

• the lateral gate opens, allowing the TMS to exit sideways into the lipid bilayer

• the rest of the protein is synthesizes in the cytosol

Insertion of a Single Pass Membrane Protein: Figure

Hydropathy Plot of Type 1 Membrane Protein

Membrane Protein Type 2

• a single-pass transmembrane protein where the N-terminus is located on the cytosolic side of the membrane and the C-terminus is on the extracellular (or luminal) side

• not made with a signal peptide

• there is a signal anchor sequence (aka start transfer sequence) near the N-terminus of the protein, which is a TMS recognized by SRP

• eg. transferrin receptor, Golgi galactosyltransferase

Insertion of Membrane Protein Type 2

• translation begins in the cytosol

• SRP binds to the emerging start transfer sequence and pauses translation

  • SRP-RNC complex docks to the SRP receptor on the ER membrane

• SRP and receptor release after GTP hydrolysis and the ribosome is handed off to Sec61, translation resumes

• the sequence is inserted into the channel as a hairpin loop

• the charge distribution around the signal anchor determines orientation

  • positive-inside rule

    • therefore N-terminus stays in cytosol

• the start-transfer sequence exits the translocon laterally thru the gate into the bilayer

• the rest of the protein continues to be synthesized in the ER lumen

Insertion of Membrane Protein Type 2: Figure

Positive-Inside Rule

• + charged residues (eg. Lys, Arg) on the cytosolic side interact with - charged phospholipid head groups of cytosolic leaflet of ER membrane

Possible explanations:

• effect of - charged phospholipids located in the inner leaflet

• in bacteria, the effect of the proton gradient (charge separation creating dipole across the membrane)

Membrane Protein Type 3

• inserted using a "signal-anchor" sequence that results in the N-terminus on the extracellular side and the C-terminus on the cytosolic side,

• lacks the cleavable N-terminal signal peptide

• there is a signal anchor sequence (aka start transfer sequence) near the N-terminus of the protein, which is a TMS recognized by SRP

• eg. cytochrome P450

Insertion of Membrane Protein Type 3: Details

• two modes of insertion are possible: NH₂ in (Type 2) or NH₂ out (Type 3)

• orientation depends on positive/negative a.a charge distribution around the start transfer sequence

  • + charges tend to remain on the cytosolic side

  • - charges are present around the translocation channel

Insertion of Membrane Protein Type 3: Steps

• translation begins in the cytosol and an internal, hydrophobic signal anchor sequence emerges. It acts as an ER targeting signal and membrane anchor

• SRP recognizes and binds this hydrophobic region, translation is paused

• SRP-RNC complex docks at SRP receptor on ER membrane

• GTP hydrolysis releases SRP, translation resums

• the C terminal of the start transfer sequence contains + charged residues, so this side stays in the cytosol

• the hydrophobic signal anchor (TMS) enters the Sec61 channel then exits laterally into the bilayer via lateral gate

Hydropathy Plot of Membrane Type 2/3

Membrane Protein Type 4

• multi-pass transmembrane proteins

• use several internal signal-anchor (start-transfer sequences) and stop-transfer sequences

• the N-terminus and C-terminus orientation depend on the number of transmembrane segments

  • even # of TMS: N and C on same sides

  • odd # of TMS: N and C on opposite sides

• the biogenesis of multi-pass membrane proteins depend on start-transfer and stop transfer sequences

  • start-transfer signals initiate translocation, which continues until reaching a stop-transfer sequence

    • subsequent start transfer sequences reinitiate translocation 

• SRP scans for the first hydrophobic segment that emerges from the ribosome

  • a similar scanning process continues until all the hydrophobic regions are inserted

Reporter Enzyme

• fused to different parts of the protein being studied, typically the N-terminus or C-terminus

• these enzymes have activity only if there are in a specific cellular compartment (eg. cytosolic vs. lumenal), so by seeing where the enzyme is active, researchers can tell which side of the membrane a part of the protein ended up on 

Reporter Enzyme: Alkaline Phosphatase

• alkaline phosphatase hydrolyzes a substrate called XP

  • XP is membrane impermeable

• when alkaline phosphatase is transported to the periplasm, XP is hydrolyzed and bacterial colonies turns blue

• when alkaline phosphatase remains in the cytosol, bacterial colonies are white