Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

PROTEIN FUNCTION
133
soluble protein with
green fluorescent tag
pre-formed FUS
protein gel
FUS
t/2 no dissociation
SOLUBLE PROTEIN
REPLACED BY BUFFER
hnRNPA2
hnRNPA1
t/2 = 10.1 min
t/2 = 3.6 min
(A)
dissociation of green protein from
gel is measured by fluorescence
microscope as a function of time
(B)
0.5 1 2 3 5 10 15 20 30 45 60
time after washing
Figure 3–35 Measuring the association between “reversible amyloids.” (A) Experimental setup. The fiber-forming domains
from proteins that contain a low-complexity domain were produced in large quantities by cloning the DNA sequence that encodes
them into an E. coli plasmid so as to allow overproduction of that domain (see p. 483). After these protein domains were purified
by affinity chromatography, a tiny droplet of concentrated solution of one of the domains (here the FUS low-complexity domain)
was deposited onto a microscope dish and allowed to gel. The gel was then soaked in a dilute solution of a fluorescently
labeled low-complexity domain from the same or a different protein, making the gel fluorescent. After replacing the dilute protein
solution with buffer, the relative strength of binding of the various domains to each other could then be measured by the decay of
fluorescence, as indicated. (B) Results. The low-complexity domain from the FUS protein binds more tightly to itself than it does
to the low-complexity domains from the proteins hnRNPA1 or hnRNPA2. A separate experiment reveals that these three different
RNA binding proteins associate by forming mixed amyloid fibrils. (Adapted from M.Kato et al., Cell 149: 753-767, 2012).
in the cell can form a hydrogel that pulls these MBoC6 and n3.318/3.30.6 other molecules into punctate
structures called intracellular bodies, or granules. Specific mRNAs can be sequestered
in such granules, where they are stored until made available by a controlled
disassembly of the core amyloid structure that holds them together.
Consider the FUS protein, an essential nuclear protein with roles in the transcription,
processing, and transport of specific mRNA molecules. Over 80 percent
of its C-terminal domain of two hundred amino acids is composed of only
four amino acids: glycine, serine, glutamine, and tyrosine. This low complexity
domain is attached to several other domains that bind to RNA molecules. At high
enough concentrations in a test tube, it forms a hydrogel that will associate with
either itself or with the low complexity domains from other proteins. As illustrated
by the experiment in Figure 3–35, although different low complexity domains
bind to each other, homotypic interactions appear to be of greatest affinity (thus,
the FUS low complexity domain binds most tightly to itself). Further experiments
reveal that that both the homotypic and the heterotypic bindings are mediated
through a β-sheet core structure forming amyloid fibrils, and that these structures
bind to other types of repeat sequences in the manner indicated in Figure 3–36.
Many of these interactions appear to be controlled by the phosphorylation of serine
side chains in the one or both of the interacting partners. However, a great
deal remains to be learned concerning these newly discovered structures and the
varied roles that they play in the cell biology of eukaryotic cells.
weak cross-beta
spine
protein with
low-complexity
domain
bound protein
binding site for
other proteins
with repeated
sequences or for
RNA molecules
Figure 3–36 One type of complex that
is formed by reversible amyloids. The
structure shown is based on the observed
interaction of RNA polymerase with a
low-complexity domain of a protein that
regulates DNA transcription. (Adapted from
I. Kwon et al., Cell 155:1049–1060, 2013.)