Abstract
The high affinity of certain cellular polyanions for many proteins (polyanion-binding proteins (PABPs)) has been demonstrated previously. It has been hypothesized that such polyanions may be involved in protein structure stabilization, stimulation of folding through chaperone-like activity, and intra- and extracellular protein transport as well as intracellular organization. The purpose of the proteomics studies reported here was to seek evidence for the idea that the nonspecific but high affinity interactions of PABPs with polyanions have a functional role in intracellular processes. Utilizing yeast protein arrays and five biotinylated cellular polyanion probes (actin, tubulin, heparin, heparan sulfate, and DNA), we identified proteins that interact with these probes and analyzed their structural and amino acid sequence requirements as well as their predicted functions in the yeast proteome. We also provide evidence for the existence of a network-like system for PABPs and their potential roles as critical hubs in intracellular behavior. This investigation takes a first step toward achieving a better understanding of the nature of polyanion-protein interactions within cells and introduces an alternative way of thinking about intracellular organization.
Highlights
As suggested by Chu et al [22] it is plausible that the common sites facilitate the access of HS to the unique sites, we propose that such common sites may represent a functional surface for nonspecific binding to polyanionic surfaces in a cell that can regulate and/or stabilize fibroblast growth factor-2 (FGF-2) or heparinbinding epidermal growth factor-like growth factor (HB-EGF) and/or align them for further interactions with other proteins
Such less specific sites on PABPs should be more available for interaction with polyanions from a statistical point of view
We showed that PABPs that are functionally important exhibit high affinity toward polyanions not because of novel sequence signatures but rather, at least partially, through nonspecific electrostatic interactions
Summary
Lyophilized G-actin from bovine muscle (molecular mass, 43 kDa), heparin and HS from porcine intestinal mucosa (molecular masses of 18 and 14 kDa, respectively), the metachromatic dye azure A, calf thymus double-stranded DNA, and dextran sulfate (average molecular mass, 5 kDa) were purchased from Sigma. Bovine brain tubulin (molecular mass, 100 kDa) was donated by Dr Richard Himes of the division of Biological Sciences at the University of Kansas. EZ-link biotin-LC-hydrazide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), MES, and EZ-link psoralen-PEO3-biotin were purchased from Pierce. Protein array kits, including buffers and controls, were obtained from Invitrogen
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