Protein transport and translocation

1
Protein transport and translocation
10-1

• Protein translocation in bacteria, eukaryotes
• targeting signals
• import, export systems bacterial, ER,
  chloroplasts, peroxisomes, mitrochondria
• nuclear import

2
Overview of protein transportand translocation
10-2

• at least 40 of all cellular proteins are
• inserted into a membrane
• translocated into an organelle, nucleus
• exported outside the cell or to the periplasm
• proteins must be kept in translocation-competent
  form (i.e., either partially or entirely unfolded
• exception is peroxisomes, nucleus
• proteins must be folded/assembled after
  translocation molecular chaperones are usually
  involved
• translocation is an energy dependent process

3
Protein translocation systems
10-3
e
e
i
i
i
e
IM, inner membrane IMS, inner membrane space P,
periplasm OM, outer membrane TL, thylakoid
lumen TM, thylakoid membrane
SecYEG, Sec61, TOM, TIM, TOC are protein subunits
of the translocation systems
adapted from Schatz and Dobberstein, Science 271,
1519 (1996)
4
Targeting signals
10-4
export signals

• blue is hydrophilic (H-phil)
• red is hydrophobic (H-phob)
• curling lines are helical
• zig-zags are turns
• OH denotes hydroxylated residues
• denotes positively charged aas
• most signals are at the N-terminus
• can be cryptic

H-phobobic
H-philic
H-phob
H-phil
H-phob
import signals
5
Translocation in bacteria
10-5

• two major pathways for translocation in
  bacteria Sec and SRP pathways
• both converge at SecYEG translocon and use SecA,
  a peripherally-bound ATPase that supplies the
  energy for translocation
• SecB binds to nascent chains containing a signal
  sequence and maintains the preprotein in
  translocation-competent form, then binds SecA
  SRP docks with membrane receptor, FtsY (simpler
  homologues of eukaryotic SRP and SRP receptor)

• archaea lack SecB, have SRP/FtsY but no SecA
  what drives translocation?
• archaeal SRP, FtsY, SecYEG more closely related
  to eukaryotic proteins (SecYEG)

6
Structure and function of SecB
10-6
7
Translocation into the ER
10-7

• Sec61 is a hetero-trimeric complex composed of
  a, b, g subunits related to SecYEG
• SRP is a ribonucleoprotein complex composed of
  7S RNA and numerous proteins
• binding of signal sequence is modulated by NAC
• SRP pathway is co-translational SRP mediates
  arrest of elongation until it docks with SRP
  receptor translocation then proceeds through
  Sec61
• SRP is the major pathway used for import into ER

• a post-translational translocation pathway that
  makes use of Sec61 also exists preproteins are
  maintained in a translocation-competent form by
  Hsp70/Hsp40

8
Folding in the Endoplasmic reticulum
10-9
9
Translocation into chloroplasts
10-9

• Toc components, mediate translocation (Toc75 is
  the translocon) it is unclear how preproteins
  are targeted to the channel Hsp70/Hsp40 may be
  involved
• Hsp70 in both the IMS and the stroma assist the
  threading of the preprotein into the chloroplast
• an Hsp100 chaperone also called ClpC (AAA
  ATPase) also binds preproteins in the stroma
• Hsp70/chaperonin (Cpn60) may assist
  folding/assembly of newly-imported protein
• import into thylakoids (used for respiration)
  uses the SRP pathway

10
Translocation into peroxisomes
10-10

• targeting of proteins is initiated
  post-translationally by Pex5/7 proteins, which
  bind the peroxisomal targeting signal (PTS)
• translocon not well defined possibility of
  vesicular budding?
• gated pore that is regulated by membrane
  proteins?
• first organelle demonstrated to import proteins
  without a PTS, by virtue of assembly with other
  proteins that contained a PTS
• various protein oligomers are imported into
  peroxisomes
• antibodies with PTS, and 9 nm gold particles
  could be imported

Other transport mechanisms likely involve folded
proteins, including the twin-arginine (Tat)
transport system of bacteria, and the
cytoplasm-to-vacuole targeting pathway of yeast
11
Translocation into mitochondria
10-11

• delivery of preproteins to mitochondria depends
  on either Hsp70/Hsp40 or MSF, mitochondrial
  import stimulation factor (MSF)
• evidence now that Hsp90 is also involved
• mtHsp70/Tim44/Mge (GrpE) is required for import
  Tim44 contains J domain
• Big debate
• brownian ratchet or pulling model for Hsp70
  system-mediated import of proteins

• protein folding following import depends on
  Hsp70, chaperonin (Hsp60)

12
Import into the nucleus
10-12

• nuclear localization signal (NLS) is typically
  highly basic e.g., the SV40 large tumor antigen
  (T ag) has the sequence PKKKRKV
• a/b1 importin hetero-dimer recognizes and binds
  the NLS (or b importin alone)
• b importin docks with NPC and mediates
  interaction with Ran (GDP form)
• directionality conferred by nature of guanine
  nucleotide bound to Ran
• Ran binding protein (RanBP) is required for b
  importin binding to RanGDP Ran GTPase activating
  protein (RanGAP) and nucleotide-exchange factor
  (RCC) are cytoplasmic and nuclear
• cytopl. RanGDP required for import nuclear
  RanGTP required for release
• conversely, RanGTP binds substrate with NES in
  the export direction
• proteins to be imported can be in a native/near
  native form

13
Structure of the nuclear pore complex
- RanGTP
bar, 50 nm
RanGTP
14
Mechanism of import into nucleus
10-14

• some nuclear pore proteins (nucleoporins)
  contain core FxFG repeats (yellow)
• b importin contains heat repeats that bind the
  FxFG repeats (Heat repeats 5, 6, 7 are shown in
  red, green and blue)
• the FxFG repeats interdigitate in grooves formed
  by the Heat repeats
• interaction of b importin with nucleoporins
  allows transport across the nuclear pore complex

Core FxFG repeats found in nucleoporins. Each
repeat is separated by a linker region
Bayliss et al. (2000) Cell 102, 99-108.
15
Heat repeat-containing protein
10-15

• 15 heat repeats of protein phosphatase 2A
• conservation is to one side of the repeat
  structure

Groves et al. (1999) Cell 96, 99-110.