soluble protein with green fuorescent tag hnRNPA2 t/2 = 10.1 minSOLUBLE PROTEIN REPLACED BY BUFFER hnRNPA1 t/2 = 3.6 min 0.5 1 2 3 5 101520304560dissociation of green protein from time after washinggel is measured by fuorescence (A) microscope as a function of time (B)
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 and 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.
protein with low-complexity domain weak cross-beta spine 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.)