Volume 24 April 1, 2013
MBoC | ARTICLE
Means of self-preservation: how an intrinsically
disordered ubiquitin-protein ligase averts
Eric K. Fredrickson, Sarah V. Clowes Candadai, Cheuk Ho Tam, and Richard G. Gardner
Department of Pharmacology, University of Washington, Seattle, WA 98195
ABSTRACT Ubiquitin-protein ligases (E3s) that ubiquitinate substrates for proteasomal deg-
radation are often in the position of ubiquitinating themselves due to interactions with a
charged ubiquitin-conjugating enzyme (E2). This can mediate the E3’s proteasomal degrada-
tion. Many E3s have evolved means to avoid autoubiquitination, including protection by
partner or substrate binding, preventative modifications, and deubiquitinating enzyme re-
versal of ubiquitination. Here we describe another adaptation for E3 self-protection discov-
ered while exploring San1, which ubiquitinates misfolded nuclear proteins in yeast for pro-
teasomal degradation. San1 is highly disordered in its substrate-binding regions N- and
C-terminal to its RING domain. In cis autoubiquitination could occur if these flexible regions
come in proximity to the E2. San1 prevents this by containing no lysines in its disordered
regions; thus the canonical residue used for ubiquitin attachment has been selectively elimi-
nated. San1’s target substrates have lost their native structures and expose hydrophobicity.
To avoid in trans autoubiquitination, San1 possesses little concentrated hydrophobicity in its
disordered regions, and thus the that feature San1 recognizes in misfolded substrates has
also been selectively eliminated. Overall the presence of key residues in San1 have been
evolutionarily minimized to avoid self-destruction either in cis or in trans. Our work expands
the ways in which E3s protect themselves from autoubiquitination.
The ability to destroy proteins with a high degree of specificity is
essential to cellular function. One means by which eukaryotes spe-
cifically degrade proteins is through the ubiquitin-proteasome
pathway, which is used typically for one of two cellular functions:
1) spatial or temporal regulation of a functional protein or
2) removal of a misfolded protein among a pool of normally folded
proteins. Ubiquitination of substrates occurs via an enzymatic cas-
cade of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating
enzyme (E2), and a ubiquitin-protein ligase (E3), which typically
confers substrate specificity within the cascade (Ciechanover,
2006). Because E3s interact with E2s charged with ubiquitin, there
is the potential for an E3 to cause its own autoubiquitination,
which can lead to the E3’s proteasomal degradation in vivo.
Unnecessary autoubiquitination and degradation would reduce
the functional levels of active E3s, and thus means of protection
must have evolved to prevent or regulate this possibility.
Four ways have been identified that minimize E3 autoubiquit-
ination and maintain E3 stability in vivo. The first is through the in-
teraction of the E3 with its complex partners. For example, interac-
tion of the yeast E3 Hrd1 with Hrd3 prevents autoubiquitination of
Hrd1 and its degradation by the proteasome (Plemper et al., 1999;
Gardner et al., 2000). The second is protection of the E3 by the
binding of a substrate. This is most notable in cullin E3s, where the
binding of a substrate to the adapter F-box protein protects the
F-box protein from ubiquitination (Galan and Peter, 1999; Li et al.,
2004). The third is through posttranslational modification of the E3.
For instance, phosphorylation or acetylation of the mammalian E3
Mdm2 decreases its autoubiquitination and degradation (Feng
et al., 2004; Wang et al., 2004). The fourth and final way is removal
of ubiquitin from the E3 by the activity of a deubiquitinating
Ramanujan S. Hegde
National Institutes of Health
Received: Nov 16, 2012
Revised: Jan 18, 2013
Accepted: Jan 23, 2013
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E12-11-0811) on January 30, 2013.
Address correspondence to: Richard Gardner (firstname.lastname@example.org).
Abbreviations used: HSV, herpes simplex virus; NLS, nuclear localization sequence.
© 2013 Fredrickson et al. This article is distributed by The American Society for Cell
Biology under license from the author(s). Two months after publication it is avail-
able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society of Cell Biology.
1042 | E. K. Fredrickson et al. Molecular Biology of the Cell
degraded in both the presence and absence of endogenous San1,
whereas wild-type San1 was stable (Figure 2A). We believed it
possible that degradation of these mutants could be independent
of the Lys residue and that an analogous conservative mutation
would be similarly sufficient to cause degradation. However, muta-
tion of the same 17 residues to either Asn or Arg, the most conser-
vative changes in relation to Lys, did not appreciably alter the stabil-
ity of San1 (Figure 2, B vs. A). Thus specific addition a single Lys
residue in San1’s highly disordered N- and C-terminal regions con-
sistently disrupted San1’s stability.
The underlying assumption of the hypothesis is that addition of
a single Lys residue in the N- and C-terminal regions should result in
degradation through in cis autoubiquitination. However, it is possi-
ble that addition of a Lys residue could also result in the recognition
of a “plus Lys” San1 mutant as a substrate by another San1 mole-
cule, leading to degradation through in trans autoubiquitination.
Therefore, it was important to distinguish between an in cis and an
in trans mode for the degradation of the “plus Lys” San1 mutants. If
degradation occurs as a result of in cis autoubiquitination, we pre-
dicted that introducing an additional RING-inactivating mutation
would result in the stability of the “plus Lys” San1 mutant even in
the presence of functional endogenous San1. Conversely, if degra-
dation occurred as a result of in trans autoubiquitination, we pre-
dicted that introducing a RING-inactivating mutation would result in
degradation of the “plus Lys” San1 mutant only when endogenous
enzyme. For example, the stability of the mammalian E3 Nrd1 is
dependent on the catalytic activity of the deubiquitinating enzyme
USP8 (Wu et al., 2004). In each case of E3 autoubiquitination, it is
not clear whether autoubiquitination is a functional means to regu-
late the E3, similar to the specific ubiquitination of a substrate, or
is simply due to the inherent activity of the E3 against itself.
In this article, we describe an additional mode of E3 self-protec-
tion that we discovered while studying the yeast E3 San1, which
mediates the ubiquitination of misfolded nuclear proteins for
proteasome-mediated degradation (Gardner et al., 2005). Our pre-
vious studies revealed that San1 possesses highly disordered N- and
C-terminal regions that contain interspersed substrate-binding sites
used for misfolded substrate recognition (Rosenbaum et al., 2011).
In particular, San1 recognizes exposed hydrophobicity within its mis-
folded substrates (Fredrickson et al., 2011). These observations led
to two questions concerning how San1 prevents its own ubiquitina-
tion and subsequent proteasome degradation. First, how does San1
prohibit its highly disordered and conformationally flexible regions
from in cis autoubiquitination if/when they become positioned near
the charged E2 bound by San1’s RING domain? Second, if San1
lacks structure, why is one San1 molecule not recognized for in trans
autoubiquitination by another San1 molecule if San1 is capable of
targeting proteins that have lost their native structures? Here we
reveal the molecular means by which San1 has evolved to prevent
its own in cis and in trans autoubiquitination and degradation.
San1 lacks Lys residues in its N- and C-terminal regions
The first clue for how San1 might be protected from in cis autoubiq-
uitination came from examining the amino acid distribution of San1’s
primary sequence. On initial inspection, we noticed that the overall
Lys content for San1 is atypical. San1 possesses only 13 Lys residues,
whereas a typical protein the size of San1 is predicted to have ∼40 Lys
residues based on average codon usage in yeast (Saccharomyces
Genome Database; www.yeastgenome.org). On closer inspection,
we found that this unusual feature could be ascribed to the fact that
San1’s highly disordered N- and C-terminal regions were devoid of
Lys residues (Figure 1A). The 13 Lys residues present in San1 are clus-
tered in or near the RING domain (Figure 1A), with nearly half com-
prising San1’s nuclear localization sequence (NLS; Figure 1A). Be-
cause E3s typically mediate the covalent attachment of ubiquitin to
the free amino group in the side chains of Lys residues in their sub-
strates, we hypothesized that the lack of Lys residues in San1’s N- and
C- terminal regions protects San1 from in cis autoubiquitination
Introduction of a single Lys residue in San1’s
N- and C-terminal regions results in rapid degradation
If a lack of Lys residues prevents San1 in cis autoubiquitination, we
predicted that the addition of a single Lys residue in San1’s N- and
C-terminal regions would disrupt San1’s stability. To test our predic-
tion, we randomly made 17 single Arg- or Asn-to-Lys point muta-
tions along the length of San1’s N- and C-terminal regions (Figure
1A, X’s). Because the N- and C- terminal regions are not completely
disordered, the mutations were made in predicted regions of order
and disorder. We then examined the stability of the “plus Lys” mu-
tant San1s in cells with the endogenous SAN1 gene intact (SAN1) or
deleted (san1∆). To monitor stability and distinguish the “plus Lys”
mutant San1s from endogenous San1, we tagged each San1 mutant
at its C-terminus with a 3× herpes simplex virus (HSV) epitope, which
does not alter the function of San1 (Gardner et al., 2005). In every
case, we found that the “plus Lys” San1 mutants were rapidly
FIGURE 1: Sequence features of San1. (A) Representation of the
overall domain topology of San1. Endogenous Lys residue positions
are marked on top. A line denotes the presence of an NLS in the
RING domain (Gardner et al., 2005). PONDR (www.pondr.com/)
evaluation of San1’s intrinsic disorder is on the bottom. Gray box
highlights the RING domain. The X’s on the 0.5 line mark the location
of the “plus Lys” mutants. (B) Model of in cis autoubiquitination upon
addition of a Lys residue in the intrinsically disordered C-terminal
region of San1.
Volume 24 April 1, 2013 How San1 prevents autoubiquitination | 1051
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We thank Makoto Kawamukai for providing plasmids and Ning
Zheng for key discussions and experimental advice on San1’s se-
quence characteristics. R.G.G. thanks Chris Hague for many illumi-
nating conversations on averting in cis and in trans degradation.
This work was supported by National Institutes of Health/National
Institute of General Medical Sciences Training Grant 5T32GM007750
(E.K.F.), National Institutes of Health/National Institute on Aging
Grant R01AG031136 (R.G.G.), an Ellison Medical Foundation
New Scholar Award in Aging (R.G.G.), and a Marian E. Smith Junior
Faculty Award (R.G.G.).
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