Membranes

Question 1

How do cell membranes form?

Answer 1

Bimolecular structures which arise spontaneously when amphipathic lipids are mixed with water.

Question 2

What are amphipathic lipids?

Answer 2

Lipids with a polar and non polar region.

Question 3

How are cell membranes stabilised?

Answer 3

By the hydrophobic effect.

Hydrophillic parts organise themselves away from the water.

Polar interactions between the hydrophillic head groups and water.

Non covalent interactions between the lipid molecules

Question 4

What does the plasma membrane also contain?

Answer 4

Protein. 52% of the yeast PM is protein.

Many of the lipids and proteins on the outer surface of the PM are glycosylated on the outside.

Question 5

Describe membrane potentials?

Answer 5

The lipid bilayer forms an effective barrier to the free diffusion of ions and charged molecules.

Lipid bilayers have a high capacitance and are capable of supporting a transmembrane voltage, rising from the impermeability.

Cytosol is more negative.

Potentials arise from the unequal distribution of charge that can occur across semi-permeable membranes. Every ion transport process contributes to the imbalance between the positive and negative charges in a membrane bound compartment.

Question 6

Why are membrane potentials important?

Answer 6

Ion transport
Energy transduction
Nerve function

Question 7

How is membrane fluidity determined?

Answer 7

Temperature:
As temperature falls, molecular motion decreases and so the phospholipids pack together more closely. Below the “transition temp” the membranes change from a fluid crystalline state into a semi rigid gel state.

Fatty acid composition:
Increasing the length of fatty acid chains reduces fluidity by increasing the number of favourable reactions between the closely packed chains.
Adding C-C double bonds increases fluidity by disrupting the close packing of the bilayer.

Question 8

Example of a peripheral protein?

Answer 8

Cytochrome C
Electron transport protein localised on the outer surface of the inner mitochondrial membrane.

Anchorage can be achieved by non covalent interactions with polar and non polar regions of membrane surfaces.

Question 9

How do membrane proteins attach?

Answer 9

Many peripheral proteins are covalently modified by adding a non-polar chain that can be inserted into the bilayer, as it tries to escape the water.

Often used to attach cytosolic proteins to membrane surfaces.

GPI anchors provide an important mechanism for attaching proteins to the external surface of the plasma membrane. Covalent modification provides an effective mechanism for localising soluble proteins on membrane surfaces

Question 10

How are GPI anchors added to proteins?

Answer 10

Glypiated (GPI-linked) proteins contain a signal sequence, thus directing them to the endoplasmic reticulum (ER).

The protein is co-translationally inserted in the ER membrane via a translocon and is attached to the ER membrane by its hydrophobic C terminus; the majority of the protein extends into the ER lumen.

The hydrophobic C-terminal sequence is then cleaved off and replaced by the GPI-anchor. As the protein processes through the secretory pathway, it is transferred via vesicles to the Golgi apparatus and finally to the plasma membrane where it remains attached to a leaflet of the cell membrane.

Since the glypiation is the sole means of attachment of such proteins to the membrane, cleavage of the group by phospholipases will result in controlled release of the protein from the membrane.

Question 11

Common motifs on integral membrane proteins?

Answer 11

1. α-helices:
   The transmembrane region of bacteriorhodopsin is made up of 7 α-helixes. Membrane spanning domains can be predicted using a hydropathy plot.
2. β-barrels:
   The transmembrane region of porin, a pore forming protein from the outer membrane of bacteria, is constructed from antiparallel β strands.
   Pore is filed with water and the amino acid side chains facing the pore are hydrophilic. The outer surface is embedded in the membrane and so is non-polar. Smaller channels with gating properties can be made from clusters of amphipathic α-helices.

Question 12

Describe membrane asymmetry?

Answer 12

Phospholipids are distributed unequally between the two surfaces.
Flip flop of the phospholipids is energetically unfavourable which helps to maintain the asymmetry.

Desirable for maintaining the cell membrane architecture. Integral membrane proteins have to span the membrane in the correct orientation.

Transverse asymmetry is observed because the orientation of lipids and proteins is controlled during the assembly and maintenance of membranes.

Phospholipid translocators (flippases) facilitate lipid movement between leaflets at the site of synthesis and elsewhere, by providing an environment for polar group to cross the membrane.

Asymmetry is maintained during vesicular transport, and exo/endocytosis. Specific mechanisms exist for directing newly synthesised proteins into particular membranes.

Membranes show lateral asymmetry which allows the formation of lipid rafts – transient micro-domains that are rich in cholesterol and glycosphingolipids.

Question 13

How does protein targeting work?

Answer 13

Newly synthesised proteins have to be directed to their correct locations.

Polypeptides synthesised by ribosomes in the cytosol are committed to one set of destinations. Polypeptides synthesised by ribosomes associated with the cell membrane or the ER are committed to another set of destinations.

1. Default pathways are followed in the absence of specific instructions
2. Signals can be encoded in the primary sequence of a protein (signal sequences) or in its tertiary structure (signal patches)
3. Further signals can be acquired by glycosylation during post-translational processing
4. Vectorial transport: the non-random transport of proteins to specific locations is driven forward by the consumption of energy

Question 14

How do proteins move from ribosomes to ER?

Answer 14

Binding of the N terminal signal sequence by a ribonucleoprotein complex called the SRP triggers the transfer and the delivery of the newly synthesised polypeptide to the ER membrane/lumen.

Question 15

How does the SRP (Signal Recognition Particle) work?

Answer 15

SRP Binds to a signal sequence at the N terminus of a protein emerging from the ribosome
and stops translation.

Transfers the stalled ribosome to a ribsome receptor on the rough endoplasmic reticulum
(ER) by binding to an adjacent SRP receptor.

Hydrolysis of GTP releases the SRP from the ribosome allowing protein synthesis to resume with concurrent transport of the protein into the ER via a peptide translocation complex in
the membrane.

Overall energetics: GTP/GDP cycle.
Free ribosome + nGTP → Bound ribosome + nGDP

Question 16

Protein translocation into the ER?

Answer 16

Protein synthesis resumes after release of the SRP, the growing polypeptide chain is threaded through the membrane into the ER lumen by an ATP-consuming process, and the signal sequence is removed by a signal peptidase.

Alternatively the nascent polypeptide can be instructed to stay in the membrane by a stop-transfer sequence and multiple sets of instructions can be used to generate membrane proteins with multiple transmembrane domains

In prokaryotes the N-terminal signal sequence directs the ribosome to the cell membrane and adding such a sequence to a cloned gene is a useful trick for exporting a heterologously expressed gene product.

Question 17

How does protein glycosylation occur?

Answer 17

Nitrogen-linked glycosylation of proteins occurs while the protein is located in the ER membrane. The oligosaccharide moiety is assembled in the cytosol and ER lumen on a hydrophobic carrier – dolichol phosphate.

The activated oligosaccharide moiety is transferred to ASP residues on the luminal side of the membrane. After further modification, the new glycoprotein is exported to the Golgi in a vesicle that buds off from the ER.

Further modification of Nitrogen-linked glycoproteins, and the synthesis of Oxygen-linked moieties occurs in the Golgi.

Signal patches on some proteins are recognised by an enzyme that phosphorylates mannose residues which then target the protein to the lysosomes.

Unmarked proteins are transported to the plasma membrane in either the lumen or membrane of secretory vesicles.

Question 18

How does protein import to mitochondria occur?

Answer 18

Most mitochondrial proteins are encoded by the nucleus and synthesised using cytosolic ribosomes.

Chaperone proteins prevent folding before translocation, allowing the nitrogen-terminal signal sequence to dock with a receptor on the outer mitochondrial membrane

After docking, the chaperone proteins are removed by an ATP dependent process

Insertion into the inner mitochondrial membrane is driven by the membrane potential and translocation is driven by ATP. On arrival in the matrix, cleavage of the first signal peptide may reveal a second signal directing the protein into the inner mitochondrial membrane.

Question 19

How does protein import to chloroplasts occur?

Answer 19

Similar to mitochondria – chaperone proteins, multiple signals may be present, and energy is required (ATP is the energy source for import into the stroma).

Also evidence that some proteins are imported into the thylakoid lumen in a fully folded state, via the ΔpH-dependent TAT pathway that is also used for the export of certain proteins into the bacterial periplasm.

Question 20

Consequences of entrusting membrane transport to proteins?

Answer 20

1. Acceleration:
   Carriers: up to several thousand ions/s
   Channels: 10,000 times faster. Close to free diffusion rate.
2. Selectivity:
   Specificity of the interactions between the protein and the transported solute.
3. Regulation:
   Channel proteins can be gated, opening and closing in response to binding (ligand gated) or to changes in membrane potential (voltage gated).
4. Coupling:
   Co transport of solutes and input of energy.

Question 21

What determines the direction of spontaneous movement across a membrane?

Answer 21

Concentration and membrane potential.

Question 22

How does the Na+K+ ATPase work?

Answer 22

1. Binding of 3 intracellular Na+
2. Phosphorylation of an Asp residue by ATP inducing a conformational change
3. Release of Na+ to extracellular space
4. Binding of 2 extracellular K+
5. Dephosphorylation reversing the previous conformational change
6. Release of K+ into the cytosol

Na+ dependent phosphorylation of the carrier and K+ dependent dephosphorylation is the key to the mechanism. This accounts for 25% of the energy consumption of a resting person.