Protein Targeting and Translocation;
Biomolecular Condensates
What Are We Interested In? (see Research for a more
detailed description of projects)
Protein
Targeting and Translocation:
The vast majority of proteins are synthesized in the
cytoplasm, and yet proteins are found in every aqueous and
membranous compartment within a given cell. Protein
targeting and transport across lipid bilayers is therefore a
fundamental process in all organisms. Up to approximately half
of the proteins in an organism's proteome are inserted into or
transported across membranes by protein translocation systems,
or translocons. These translocons are typically
elaborate molecular structures that facilitate the transport
of desirable molecules while at the same time maintaining the
membrane's function as a permeability barrier. While
there are some similarities, protein translocation systems
have surprisingly varied properties such as energetic and
substrate requirements, recognition and gating mechanisms, and
pore structure. Substantial biochemical work has
provided a wealth of information about the signaling
information, the protein substrates and transport cofactors,
the energetic requirements and a general picture of the
interplay amongst all these items for many protein transport
systems. In many cases, structural studies are providing
an emerging detailed picture of the molecular arrangement of
protein components. However, there exists very little
molecular detail of the specific interactions or the kinetic
transitions between sub-states required for transport.
We are currently working on two protein transport systems that
translocate folded proteins:
The Nuclear Pore Complex
- The nuclear pore complex (NPC) spanning the
double-membrane nuclear envelope fulfills the essential
physiological role of facilitating and regulating the
extensive trafficking between the cytoplasm and nucleoplasm
in eukaryotic cells. It is unique among protein
transporters because it regulates the trafficking of diverse
substrates in both directions and it can accommodate the
passage of molecules as large as ribosomal subunits.
Pre-ribosomal subunits, mRNAs, and tRNAs are exported to the
cytoplasm. Proteins imported into the nucleus include
histones, DNA and RNA polymerases, RNA processing proteins,
ribosomal proteins, and numerous transcription
factors. Current work is focused on using
three-dimensional (3D) super-resolution single molecule
fluorescence microscopy and single particle tracking
techniques to explore different transport pathways and to
determine the extent of their structural and functional
intersections.
The Tat Machinery -
Many bacteria contain two very different general protein
translocation machineries within the cytoplasmic membrane
responsible for secretion from the cytoplasm to the
periplasm. The Sec machinery (general secretory
pathway) translocates polypeptides in an unfolded, linear
configuration energetically driven by ATP hydrolysis and the
proton motive force, or pmf (consisting of pH and electric
field gradients). In contrast, the Tat machinery
recognizes and translocates fully-assembled multi-subunit
redox proteins in the presence of the pmf alone. The
Tat system is responsible for the export of numerous
proteins important for bacterial virulence in humans, and
its absence often leads to growth defects. Since
animals, including humans, do not contain homologs of Tat
machinery proteins, inhibitors of Tat transport could
potentially find use as novel antibiotics. Current
work is focused on using inverted membrane vesicles as
individual reaction chambers to determine the functional
consequences of variable Tat stoichiometries and membrane
potentials, and to investigates the dynamics of conformation
changes.
Biomolecular Condensates/Membrane-less Organelles:
Membrane-less organelles (MLOs) are liquid-like compartments
within cells typically characterized by high concentrations of
intrinsically disordered proteins (IDPs) and RNA molecules.
Over 50 membrane-less compartments have been identified -
examples include nucleoli, stress granules, and P
bodies. These biomolecular condensates (BMCs) are
generally agreed to arise from liquid-liquid phase separation
(LLPS). LLPS is the spontaneous partitioning into dense
and dilute phases due to favorable interactions between the
separating molecules. We are currently working on the
following model protein:
FUS (fused in sarcoma)
- FUS is a nuclear DNA/RNA binding protein that regulates
transcription, splicing, and mRNA transport. It is a
major aggregating protein in amyotrophic lateral sclerosis
(ALS) and frontotemporal dementia (FTD). Arginine
methylation defects and disease-causing mutations in FUS
result in its accumulation in cytosolic MLOs called stress
granules. Solid FUS-containing inclusions are observed
in the neuronal and glial cells of ALS and FTD
patients. In vitro, FUS undergoes LLPS forming
spherical droplets, which convert into solid (typically fibrous) aggregates
over time. Current work is focused on characterizing
the physicochemical properties of FUS droplets during phase
maturation using various single molecule fluorescence
methods.