Download Folding quality control in the export of proteins by the

“Folding quality control in
the export of proteins by the
bacterial twin-arginine
translocation pathway”
DeLisa MP, Tullman D, Georgiou G.
Proc Natl Acad Sci. 13;100(10):6115-20.
Geoffrey Meyerson
Tarak Shah
October 13, 2004
Microbiology & Biotechnology
Question of the Day

What are the two known
prerequisites necessary for the
translocation of a protein through the
Tat pathway?
Agenda

Introduction & background
• Sec pathway
• Tat pathway



Overview of experiment
Discussion
Applications to biotechnology
Sec and Tat Pathways
Robinson and Bolhuis, Nature, May 2001
Recognition Sequences

Tat and Sec have
similar peptide
recognition sequences
• Both have positively
charged N-terminus
• Followed by a region of
hydrophobic amino
acids
• More polar C-terminus
before cleavage site
(leading to the rest of
the active protein)



Tat pathway contains
an arginine-arginine
motif surrounded by a
mostly conserved
peptide sequence
Sec does not contain
arginine-arginine
sequence
Typically Tat exhibits a
less hydrophobic
region than Sec
Peptide Signal Sequences
Sec Signal Sequence
Hydrophobic region
N-domain
H-domain
Tat Signal Sequence
C-domain
Hydrophobic region
Pohlschröder, FEMS Microbiology Review, September 2003
Note: Big arrows designate signal peptidase cleavage sites.
Green Fluorescent Protein
GFP formed
in cytoplasm
Translocated
through Tat
GFP formed
in cytoplasm
Not translocated
Robinson and Bolhuis, Nature, May 2001
The Tat Pathway



What is it?
How does it function?
Tat vs. Sec?
Membrane
Robinson and Bolhuis, Nature, May 2001
Leadup to Tat

Clark and Theg (1997)
• unfolding is not a
ubiquitous
requirement for
protein translocation
• at least some
domains of targeted
proteins can maintain
a nonlinear structure
during their
translocation into and
within chloroplasts

Hynds et al (1998)
• Unfolded proteins can
be translocated either
through the Sec or
Tat pathway
• “Correct folding is not
a prerequisite for
translocation”
The Experiment


Objective: To examine relationship
between protein folding and export
competence
How It Was Accomplished: By
analyzing subcellular localization of
proteins with structural disulfide
bonds in the context of oxidizing and
reducing cytoplasm and periplasm
Main Concept



Proteins do not form
disulfide bonds in a
reducing cytoplasm thus
inhibiting Tat pathway
Tat pathway was utilized
in oxidizing cytoplasm and
reducing periplasm
In oxidizing cytoplasm
and periplasm, no Tat
transport occurs
Wild type
Experiment
Control
The Experiment



Fuse to AP 8 leader peptides that
direct export via Tat pathway
Treat cell samples to prevent the
formation of disulfide bonds during
fractionation by osmotic shock
Determine the AP enzymatic
activities in the cytoplasmic and
periplasmic fractions
Western Blotting
WT
Experiment Tat mutant
Periplasm
Cytoplasm
Note:
Grey = periplasm
White = cytoplasm
Trypsin Resistance
Note: periplasmic fraction
Tat Export of FAB


FAB fusion of heavy
chain and a leader
peptide recognized by
the Tat pathway led to
tranlocation of entire
antibody
Hitchhiker mode of
export allows a
leaderless polypeptide
to be exported via
association with a
second polypeptide
that can engage Tat
Tat Leader Sequences
Tat sequence
}
}
Class I
Class II
}
}
Class I
Class II
Discussion

Tat export requires specific Tat leader
peptides as well as folding of the protein
in the cytoplasm
• ONLY folded proteins allowed to be exported
through Tat


Class I leader peptides required use of Tat
pathway
Class II leader peptides were pathway
agnostic
• The state of the protein (folding) determined
which pathway was used
Discussion


Inability to export AP and FAB
proteins in the absence of disulfide
bonds indicates the existence of a
quality control mechanism
A cytoplasmic domain of Tat can
function as a chaperone to the Tat
membrane site
Biotechnology Applications




Proteins of interest could be fused with a
Tat leader sequence.
Proteins that would normally remain
folded in the cytoplasm can now be
induced to translocate through the Tat
pathway.
This facilitates protein separation, the
most costly step in bioreactor processes
Way to engineer proteins for transport
without needed to change structure of
protein