Protein transport 5

Question 1

TAT pathway

Answer 1

• Both 3 domains: polar NTD, hydrophobic central, polar CTD
• N terminal = SRRXFLK
• C terminal = basic or uncharged
• H region = ↑ hydrobic in sec than TAT
• Sec threads unfolded proteins, Tat folded

Question 2

TAT mechanism

Answer 2

• Sec diameter - 12A, tat substrate = folded protein, hole = 70A how to seal
• Cavity diameter varies by changing number of subunits
• Or, TAT x span the whole membrane, causes membrane distortion

Question 3

Import of nuclear-encoded gene products

Answer 3

• Almost all proteins made in cytoplasm (out → in)
• Opposite for ancestral bacteria, diff mechanism
• Cytosolic chaperones guide precursor proteins to central mit entry gate

Question 4

TOM/TIM23 pathway

Answer 4

• Mediates transport from cytosol to matrix
• Amino terminal signal peptides used
• Proteins translocated by TIM23 in ΔY-dependent manner
• ATP-driven PAM needed
• MPP removes presequence

Question 5

TOM/TIM22

Answer 5

• Integrates multi-spanning IM proteins inc. mit carrier proteins
• Uses complex internal targeting signals
• Guided by small Tim proteins across iM space

Question 6

TOM/SAM

Answer 6

• Precursors of B-barrel protein of OM transferred from TOM to SAM w/ small Tim
• Homologous to Bam
• Uses short consensus in final B-strand

Question 7

Energisation of mitochondrial protein import

Answer 7

1. protein import = PT, chaperons maintain in unfolded state, release hsp70 needs ATP hydrolysis
2. ΔY of proton electrochemical gradient across IM drives movement of signal
3. Mit hsp70 binds part of polypeptide as exposed in matrix, pulls protein, ATP hydryolsis to remove

Question 8

Mechanism of pulling

Answer 8

1. Thermal ratchet
   Emerging chain slides back + forth in TIM23 by thermal motion, each time portion exposed hsp70 binds, prevents backsliding
2. Cross bridge ratchet
   Hsp70 binds to emerging polypeptide chain undergoes a conf Δ driven by ATP hydrolysis, actively pulls chain into matrix

Both = hsp70 closely assoc w/ TIM23 + hsp70 acts as ratchet

Question 9

Chloroplast import of nuclear-encoded protein

Answer 9

• Need transport molecule
  1. Cytoplasmic preprotein binds surface of chloroplast by interaction w/ intrinsic transit sequnce
  2. Protein inserted through TOC + TIC
  3. Requires GTP hydrolysis by TOC + ATP-dependent chaperon
  4. Stromal chaperone Clpc + Cpn60 bind preprotein

Question 10

LHCP

Answer 10

• LHCP = targeted to thylakoid by cpSRP-dependent transport
• Imported post-translationally into chloroplasts via TOL/TIC translocon in chloroplast envelope
• In stroma, LHCP bound by cpSRP chaperons, otherwise insoluble