Lecture 9 - how the prokaryotic cell is made

Question 1

What is the structure of the gram negative cell?

Answer 1

-outer membrane
-periplasm (in the middle of the two membranes, aqeous region of the cell contains enxymes and cell wall)
-inner membrane
-2 lipid bilayer membranes
-high proportion of cellular proteins assocaited with the evelope (~35% in E.coli)
-cytoplasm is highly packed with proteins
-

Question 2

What are the features of membrane lipids?

Answer 2

• phospholipids
• made inside the cytoplasm and need to get across
• hydrophobic tails of lipids coalesce spontaneously to exclude water (more energetically favourable to pack together to exclude extracellular components)
• head groups on the inside are different to those on the outside (asymmetric lipid composition between the two leflets in the bilayer)
• ‘lipid flippase’ (MsbA) moves phospholipids from the inner to the outer leaflet of the inner membrane (energy is required [ATP] as moving a polar group across a hydrophobic membrane)

Question 3

Why can the growth of a cells membrane NOT be spontaenous as the cell grows?

Answer 3

• if spontaenous there would result in too much lipid on the inner membrane and the membrane would collapse
• MsdA mutants lack membrane integrity, membrane vesicles keep getting released

Question 4

How was lipid asymmetry identified?

Answer 4

• take cell and treat with lysozyme to break down the cell wall bonds between peptidoglycan units
• causes the formation of spheroblasts (made up of the inner membrane and the cytoplasm)
• remove lipid head groups with phospholipase treatment and analyse by cytometry OR treat spheroblast under pressure forming inside out vesicles then treat with phospholipase and analyse

Question 5

What is the mechanism of protein assembly in the periplasm?

Answer 5

• proteins destined for the aqeous environment outside the cytoplasmic membrane contain a predictable signal sequence at the N terminus
• (+) charged amino acids followed by hydrophobic amino acids, followed by a small amino acid side chain
• although the peptide typically folds into the mature protein immediately following translation this is prevented for periplasmic protein by SecA
• SS is recognised by SecA (+SecB)
• SecA bind SS and delivers the protein to secYEG translocon
• this drives the unfoldeed peptide through the translocon (ATP-dependent)
• proteins gets threaded through and the signal peptidase cleaves the signal sequence
• this allows the protein to fold up in the correct corfmination

Question 6

Can we predict and measure localisation to the perimplasm?

Answer 6

Through bioinformatics
-look at the sequence of proteins and run it through a programme to detect whether there might be a signal peptide

Experimental confirmation: separate cytoplasm from the periplasm with lysozyme, then isolate the periplasm from the spheroblast, anaylse mature protein with mass spec

Question 7

Through what mechanisms can folded proteins be transferred to the periplams?

Answer 7

• via the Tat (twin arginine translocation) pathway
• TatA/TatB recognises signal sequences of TAT proteins (2 adjacent arginines) at the beginning of the signal peptide
• TatA/TatB picks up the protein of interest and forms a complex with TatC, protein is then passaged trough the translocon into the periplasm and the signal peptide cleaved off
• results in a fully formed protein being inserted into the periplam

Question 8

What advantages are there to pushing out a fully folded protein into the periplasm rather than an unfolded protein?

Answer 8

-may have complicated cofactor that will also need to be transported into the periplasm

Question 9

What advatages are there to pushing out an unfolded protein into the periplasm rather than a folded protein?

Answer 9

• eaier to do
• fully folded would require a large pore, meaning that large molecules and many ions could escape through
• less energetically favourable
• fully folded may have activity where not wanted

Question 10

What are the features of assembly of proteins into the innner membrane?

Answer 10

• inner membrane spans typically α-helical and consist of ~20 hydrophobic amino acid side chains
• membrane spanning regions are recognised by ribonucleoprotein complex, the signal recognition particle (SRP)
• SRP delivers peptide to the SecYEG for the assembly of the protein into the membrane
• process involved in two distinct assembly processes

Question 11

How can membrane protein localisation be predicted?

Answer 11

• Hydropathy plots
• measures how hydrophobic are the amino acids in a peptide sequence
• spans of 20 amino acids or more with high hydrophpobicity index indicated membrane localisation
• e.g. Lac permease
• 12 transmembrane helicies
• non transmembrane regions more hydrophillic and so stop in the membrane and are stable

Question 12

What is the process of assembly of proteins to the outer membrane, e.g. with porin protein

Answer 12

-outer membrane porin proteins have an inportant role as channels of low molecular weight substrates for the cell
-porin peptides are translocated initially to the periplasm via the Sec (A, YEG) system
How know if insertion into the outer membrane is spontaenous?
-consider the rate of folding into vesicles in vitro verses in viv rates
-if specifically folds into OM
-roles of LPS/periplasmic and other membrane chaperones

Question 13

How is the cell wall made? (connecting peptidoglycan)

Answer 13

• polymer of N-acetyl glucosamine/N-acetyl muramic acid/pentapeptide is inserted into the pre-existing wall structure
• bonds broken by autolysins
• bonds made by transglycosylases, transpeptidases
• activated NAG-NAM peptide units are shuttled across the membrane by lipid attachment via bactroprenol

Question 14

What is the process of translocation of proteins to outside the cell? (secretion systems)

Answer 14

• bacterial cell may be capable of delivering proteins to the outside of the cell, or directly into another cell of a different species
• at least six different systems have evolved for the translocation of proteins from the cytoplasm to the outside of the cell
• can export proteins to interfere with signalling processes in the mammalian cell -e.g. for cytoskeleton remodelling
• e.g. Type 3 secretion system (T3SS), found in many pathogenic bacteria, required for virulence
• the T3SS molecular syringe is able to cross two bacterial membranes and the membrane of a host cell, to transfer proteins from one cytoplasm to another
• e.g. into mammalian host cell
• basal body spans the inner and outer membranes of the attacker
• outer syringe (needle) extends all the way to the host cell
• inner diameter of ~20A