Lecture 6 Import to mitochondria, chloroplasts, peroxisomes and nuclei

1
Lecture 6 Import to mitochondria, chloroplasts,
peroxisomes and nuclei

• We shall see that
• 1) Import into these organelles is mainly or
  wholly post-translation
• 2) In all cases except for peroxisomes the
  proteins must be routed to the right
  sub-compartment
• 3) The structures controlling import are simple
  in peroxisomes, complex in mitochondria and
  chloroplasts and very sophisticated in the case
  of the nuclear pore

2
Mitochondrial and chloroplast subcompartments

• 1) The outer membrane
• 2) The inter membrane space
• 3) The inner membrane
• 4) The matrix
• and in chloroplasts also
• 5) The thylakoid membrane
• 6) The lumen of the thylakoid

3
Recognition of proteins destined for mitochondria

• This involves both terminal signal sequences and
  internal signal patches
• In some cases proteins for import are packaged
  with chaperones, especially Hsp70, but in other
  cases it would appear that the fully-folded
  protein is imported intact.
• There are two major recognition sites on the
  outer membrane, one recognising fully folded
  proteins, the other proteins associated with
  chaperones

4
Entry of proteins into mitochondria, general
principles
5
Targetting to mitochondria
Targetting of proteins to mitochondria involves
both internal and terminal signal sequences and
signal patches
6
Import into mitochondria
7
Transfer across the Outer membrane

• This involves the TOM (Transport - Outer
  Membrane) complex which is comprised of at least
  8 proteins
• There appear to be three methods for recognising
  proteins destined for import.
• Some proteins bind to a TOM20/22 which in turn
  binds to the universal transporter TOM40
• Others appear require binding to an inner
  membrane protein OXA1-details under dispute
• Finally yet others bind to TOM70 which presents
  them to TOM40

8
Continued

• Transfer across the outer membrane does not
  require ATP hydrolysis directly but obviously
  energy is needed as the proteins are moving up a
  concentration gradient. The energy is probably
  provided by ATP hydrolysis in binding and
  releasing Hsp70
• The transfer of proteins destined for the matrix
  is halted. This may occur soon after the outer
  membrane is contacted (the so called cis site)
  but the more important trans site halts
  translation leaving a length of amino acids
  projecting into the intermembrane space

9
The Transfer - Inner membrane complex

• This again is made up of a number of peptides.
  This complex performs a number of functions
• 1) It must recognise the signal on peptides
  projecting from the TOM complex
• 2) It must guide these through the inner
  membrane and then pull the rest of the molecule
  through.
• The matrix targetting signal must now be removed.
  This is carried out by a special Matrix
  processing peptidase, sometimes assisted by a
  second enzyme, the mitochondrial intermediate
  peptidase

10
Mechanics of Movement
11
Contimued

• Movement of proteins through the pore depends on
  the maintenance of the potential difference
  across the membrane. This is normally app. 200
  mV which is equivalent to 400,000 V/cm
• The key role in dragging the remainder of the
  proteins through the membrane is played by the
  mitochondrial form of Hsc70. Transfer requires
  ATP hydrolysis. Three mechanisms have been
  suggested. The ratchet mechanism proposes that
  transfer is by Brownian motion with Hsc70 binding
  preventing back movement. The molecular motor
  model proposes that the conformational change of
  mtHsc70 results in the protein being pulled
  across the membrane

12
TOMs and TIMs

• It is clear that there is no permanent conncetion
  between the two complexes. It is less clear
  whether there are temporary around the transfer
  pore connections or whether the sole junction is
  by the protein in transit. The inner membrane
  protein TIM54 has a projection which may reach
  the inner membrane
• It is unclear whether there are permanent contact
  sites between the inner and outer membrane or
  whether in real life the outer membranes lie
  close to the outermost parts of the inner
  membrane

13
Supply of the intermembrane space
14
Further processing

• Proteins are transferred across the TOM/TIM
  system as extended chains. A variety of
  chaperone proteins assist in the correct folding
  following transfer to the matrix
• Chaperones also assist in the assembly of inner
  membrane components which contain both
  polypeptides specified by nuclear genes and
  polypeptides specified by mitochondrial DNA

15
Other pathways

• Little is known about the insertion of outer
  membrane proteins. The most abundant protein is
  porin which cinsists almost entirely of
  transmembrane beta sheets
• Apocytochrome C appears to transverse the outer
  membrane by a special reversible pathway.
  Conversion to cytocrome C makes the process
  irreversible.
• Lactate dehydrogenase has two targetting sequnces
  - a matrix targetting sequence and a sequence
  which specifies the intermembrane space. It is
  not clear whether there is sequential transfer or
  the second signal acts as a stop transfer

16
Chloroplasts

• Transfer in chloroplasts is very similar to
  transfer in mitochondia. Thylakoid proteins
  carry two signal sequences, one specifies matrix,
  the second thylakoid

17
Import of Proteins into Peroxisomes

• Peroxisomes are a type of microbody. Microbodies
  are cell organelles bounded by a single membrane
  and are used for a variety if different
  processes. For example peroxisomes contain
  enzymes which produce hydrogen peroxide (and have
  the means for destroying it). In addition plants
  have glyoxysomes which contain the enzymes of the
  glyoxylate cycle and yeasts have a variety of
  microbodies including ones involved in methanol
  oxidation.

18
(No Transcript)
19
Targeting to peroxisomes

• Fortunately this is nice and simple. Two major
  targetting signals Pex5 (PTS1R) and PTS2R have
  been identified. The Pex5 signal is a
  carboxy-terminal tripeptide SKL.
• The mechanism of Prx5appears to involve binding
  of the SKL sequence to Pex5. This then interacts
  with a peroxisomal membrane protein called Pex14
  forming a channel. It is not clear whether Pex5
  and the protein move together across the channel
  or whether the imported protein is pushed
  through.
• In the peroxisome the signal sequences are
  removed and Pex5 is recycled with the help of
  Pex2, 10, and 12.

20
To be noted

• ATP hydrolysis is required for import
• Import into peroxisomes does not require
  unfolding of the protein chain - even gold
  particles conjugated to a peroxisomal protein are
  imported.
• Hsp70 is however needed and becomes bound to the
  exterior of the peroxisome.
• .Study of patients with Zellwegers syndrome,
  where import of proteins into peroxisomes shows
  firstly that the peroxisomal membrane proteins
  are incorporated by another route and secondly
  that at least 8 polypeptides, including Pex2, are
  involved in the transfer.

21
Peroxisome matrix and membrane proteins enter by
different routes
22
The Nucleus

• The outer nuclear membrane is continuous with the
  ER but the inner nuclear membrane, the nuclear
  lamina and the chromatin need to be supplied with
  proteins. The nuclear pore complex is a
  sophisticated gateway

23
The nuclear pore complex
24
The nuclear pore complex

• The nuclear pore complex allows large complexes
  such as ribosomes to pass through while retaining
  small peptides within the nucleus
• The complex may consist of up to 100 polypeptide
  chains
• The complex consists of two rings of particles
  with 8 spokes extending towards a central 26 nm
  pore

25
Import of Proteins into the nucleus

• This is specified by a number of signal sequences
  most of which are internal
• These signals are recognised by families of
  cytosolic receptors. These may act as
  chaperones.
• A small GTP-binding protein called ran plays a
  central role
• As with other small GTP-binding proteins a key
  role is played by the GTP/GDP exchange factor
  (GEF) and the GTPase activiating protein (GAP).
  Ran binding protein assists in GTP hydrolysis

26
The Ran cycle
27
Import and export
28
The mechanics of import

• The protein to be transported binds to an import
  receptor and moves in to the nucleus.
• Ran-GTP binds to the import receptor releasing
  the cargo
• The receptorRanGTP complex moves out of the
  nucleus
• The GTP is hydrolysed
• RanGDP is released and returns to the nucleus
  leaving the receptor free to bind another protein
• Ran GEF catalyses then exchange of GTP for GDP

29
And of export

• The empty export receptor enters the nucleus and
  binds the cargo protein and RanGTP.
• This passes out of the nucleus
• In the cytoplasm GTP is hydrolysed and GDPRan
  and the cargo protein are released
• GPDRan and the esport receptor return separately
  to the nucleus.
• The GDP bound to Ran is exchanged for a GTP

30
References

• The basic pathways for import of proteins into
  mitochondria have been understood for some time
  but the details keep changing. Several of the
  diagrams come from Lodish 5th Edn where the
  pictures are on the web. In www.whfreeman.com
• More on Ran may be found in Azuma and Dasso, COCB
  12 (2000)301-307
• Ryan and Wentes review of the Nuclear pore
  complex ) COCB 12 (2000) 361-371is almost
  unreadable but is well referenced