國立臺灣大學生命科學院動物學研究所 碩士論文
Institute of Zoology College of Life Science
National Taiwan University Master Thesis
泛素化在細胞自噬分解酵母菌胞內蛋白質中 所扮演的角色
Role of ubiquitylation in autophagic degradation of cytosolic soluble protein in Saccharomyces cerevisiae
劉昂宇 Ang-Yu Liu
指導教授：黃偉邦 博士 Advisor: Wei-Pang Huang, Ph.D.
誌謝
兩年瞬間就結束了。最先要感謝的是黃偉邦老師，收留並提供我實驗室各項
資源，讓我能盡情的享受做實驗的各種喜怒哀樂。另外，感謝陳俊宏老師、李心
予老師還有朱家瑩老師，你們百忙之中仍抽空替我口試，並包容了我的不成熟而
好心的給了我忠告，我會改進的。也要感謝植虹，在日常生活的苦悶之餘帶給大
家無盡的笑點，實在是實驗室的開心果。瑩蓉，感謝妳傾聽我內心的感觸，無形
中讓我卸下內心的壓力，能夠繼續走下去。乃彧，我一直很佩服妳能在慵懶的背
後，把事情處理得那麼有條不紊，雖然改造自己很困難，但我一直把妳當作學習
的對象呢。立恩跟鈺棋，我知道你們要顧好自己的課業，又要完成實驗進度是真
的很累，但請你們一定要加油。晉瑋學姊，就是有妳在，實驗室才能平和的度過
每一天，希望妳跟老師還有小寶寶，能幸福平靜的度過每一天。享恩學姊，有妳
傳承實驗室的各項技術，才能讓我們順利的完成研究，感謝妳啦!黃小天跟郡君，
妳們座位就在我旁邊，一定常受到我的干擾吧?真的很抱歉，感謝妳們的包容。
另外，如果沒有瑞昕、石寶跟季侑，我的研究大概也沒辦法寫成論文，要感謝你
們不厭其煩的解決我的瑣碎問題。俊彥學長、綜遠學長，每次遇到你們的熱情招
呼，都讓我精神一振…還有在台大的各位，千翔、亞凡、俐君、Milky、俐萱、豬
排等等，謝謝你們支持了我的生活，形塑了我離開台南後的性格，並且提供了我
以前想都不敢想的珍貴經歷。
最後，感謝我的爸媽，還有我的姊姊。我的一切，都是你們提供的。
CONTENTS
摘要
………..iABSTRACT
………iiINTRODUCTION
Overview of the UPS pathway………1Overview of autophagy……….……….5
The selectivity of autophagy………..9
MATERIALS AND METHODS
Strains and Media……….13Plasmid construction………13
Western blot analysis………15
Fluorescent microscopy analysis………..16
Inhibition of proteasome activity with the treatment of MG132………...16
RESULTS
Ubiquitylation does not trigger protein aggregation in yeast………...17Ubiquitylated EGFP is a substrate for autophagic degradation………20
Proteasomes, but not autophagy, degrade most ubiquitylated EGFP during starvation………..21
Autophagic degradation of UbG76V-EGFP was slower than that of
UbI44A,G76V-EGFP……….23
Low level of ubiquitylated EGFP is not a substrate for autophagic degradation….26 Delay of ubiquitylated EGFP in autophagic degradation is not unique to specific genetic background……….27
Autophagic degradation of ubiquitylated EGFP is slower than that of other cytosolic proteins………...28
DISCUSSION
Ubiquitylation triggers no protein aggregation in yeast………...32Delay, rather than acceleration, of UbG76V-EGFP in autophagic degradation……..32
Low level of ubiquitylated EGFP is not a substrate for autophagy………..34
I44A mutation destroys the delay of ubiquitylation in autophagic degradation of cytosolic protein………...34
The competition between proteasome and autophagy for UbG76V-EGFP as a substrate………35
The first report that ubiquitylation hinders autophagic degradation of cytosolic soluble proteins………38
REFERENCES
………41TABLES
………..52FIGURES
………53摘要
。 泛素是種小而保守的蛋白質，普遍的被用來標定在真核生物中將要被蛋白脢
體分解的異常構型蛋白質。細胞自噬則是真核生物中另一條降解蛋白質的路徑；
透過由雙層膜包裹的細胞質所形成的自噬小體與細胞中的液胞或溶小體進行融
合，細胞能有效的分解胞器以及蛋白質，以獲得養分應付充滿壓力的環境。已知
在哺乳動物細胞中，被泛素所標定的蛋白質除了透過蛋白脢體分解外，也會形成
蛋白質聚集體，堆積在細胞質當中，而這些蛋白質聚集體，已被證實能選擇性的
透過細胞自噬所清除。這次研究，我們發現在酵母菌中泛素修飾並不會促進蛋白
質形成聚集體，且泛素化的修飾不但不會促進蛋白質透過細胞自噬分解，反而扮
演著抑制性的角色。我們同時發現，泛素的一個已知的突變能破壞泛素與大部分
泛素結合區的交互作用，而這種泛素突變也喪失抑制細胞自噬分解蛋白質的作用
我們認為，透過泛素與某種未知蛋白質的交互作用，阻礙了泛素化蛋白質被自噬
小體包裹的過程。這個發現，是泛素抑制細胞自噬分解作用的首件案例。
Abstract
Ubiquitin is a small, conserved molecule among eukaryotes that serves as a tag
for the breakdown of misfolded-proteins by the 26s proteasome in eukaryotes.
Autophagy is another protein turnover process, which sequesters cytoplasm into
double membrane vesicles, called autophagosomes. Subsequent fusion with the
vacuole/lysosome mediates breakdown of proteins or organelles in eukaryotes facing
stressful environments. In mammalian cells, ubiquitylated protein aggregates in
cytosol associated with neural degeneration diseases were shown to be specific
substrates for autophagic degradation. However, in this study, we showed that the
ubiquitin modification does not trigger the formation of protein aggregates in
saccharomyces cerevisiae. Moreover, we found that ubiquitylation impedes, instead
promotes, the degradation of cytosolic proteins by starvation-induced autophagy in
yeast. We also identified an ubiquitin mutant, which was previously shown defective
in interacting with most known ubiquitin-binding domains (UBD), lost the delay on
autophagic degradation of cytosolic proteins. We propose that the interaction of
ubiquitin with an unknown factor prevents sequestration of ubiquitylated cytosolic
soluble proteins into autophagosomes for degradation. This is the first report
indicating that ubiquitylation of cargo proteins hinder their autophagic degradation.
Introduction
Protein turnover plays a critical role in cell survival. In eukaryotic cells, proteins are
either synthesized on ribosomes soluble in cytoplasm or attached to the endoplasmic
reticulum(ER), where they are properly modified and fold into active conformation by
ER-resident modifying enzymes and chaperones. Misfolded proteins are destined for
degradation, which protects cells from cytotoxicity and releases free amino acids for
further protein synthesis. Two major pathways were found in eukaryotes to
breakdown intracellular proteins. First is the ubiquitin-proteasome system (UPS)
pathway, in which proteins are typically modified with ubiquitins and degraded via
the proteasome (Johnson et al., 1992; Johnson et al., 1995; Pickart, 2001). The other
is the massive degradation through autophagy, in which a portion of cytoplasm was
sequestered and degraded by the vacuole in yeast or lysosomes in mammalian cells
(Tsukada and Ohsumi, 1993; Levine and Klionsky, 2004). Both pathways serve to
protect cells from cytotoxocity of misfolded proteins.
Overview of the UPS pathway
Ubiquitin is a highly conserved small protein that expresses ubiquitously and serves
multiple functions in eukaryotes (Hershko et al., 2000; Chen and Sun, 2009). It was
first described to participate in proteolysis by labeling substrate proteins to facilitate
their recognition by and degradation through the 26S proteasome. This proteolysis
pathway is termed the UPS system (Hough et al., 1986; Johnson et al., 1992; Johnson
et al., 1995; Pickart, 2001). Ubiquitin was later shown to play multiple roles in
regulation of multiple cellular processes, including endocytosis, signal transduction in
immune system, and DNA repair (Chen and Sun, 2009). Conjugation of ubiquitin to
target proteins is a complex process. Ubiquitin is first synthesized as an inactive
precursor and subsequently processed by deubiquitinatin enzymes (DUBs) into the
mature form that exposes its 76th glycine residue at the C-terminus as the active site
for the conjugation reaction. Next, for conjugation of ubiquitin to substrate proteins,
ubiquitin must be sequentially processed by E1, E2 and E3 enzymes. First, ubiquitin
is adenylated, which is catalyzed by the E1 enzymes using ATP as a substrate. The
newly formed high energy bond between ubiquitin and AMP is soon attacked by a
cystein residue at the active site of the E1 enzymes, resulting in the formation of a
thioester bond between ubiquitin and the E1 enzymes. Next, Ubiquitin is passed to a
cystein residue at the active site of the E2 enzymes, then finally to substrate proteins
forming a isopeptide stable linkage bwtween the 76th glycine of ubiquitin molecule
and the ε-amino group of a lysine residue on target proteins. This latter reaction is
catalyzed by the E3 enzymes (Kerscher et al., 2006). Based on different structures
discovered in the catalytic domains of E3 enzymes, the E3 enzymes could be further
divided into the HECT class and the RING class (Kerscher et al., 2006). The HECT
E3 enzymes, harboring an active cystein residue on the catalytic domain, form a
thioester bond with ubiquitin molecule passing from E2 enzymes before conjugation
of ubiquitin to target proteins. On the other hand, the RING E3 enzymes, which have
no cystein group on the catalytic domain, harbor a RING motif that coordinates a pair
of zinc ions. The RING E3 enzymes serve, at least in part, as scaffolds that bind
thioester bond-linked ubiquitin E2 and target proteins, facilitating the direct transfer
of ubiquitin from the E2 enzymes to target proteins. It was also shown that the RING
E3 enzymes trigger subtle conformational changes in the bound E2 enzymes,
stimulating ubiquitin release from the E2 enzymes and transfer to substrate proteins.
Due to the presence of seven lysine residues, ubiquitin could itself serves as a
substrate for ubiquitylation, leading to the formation of polyubiquitin
chian-modification on substrate proteins. The polyubiquitin chains, which are
recognized by ubiquitin-interacting proteins for downstream signaling, serve multiple
functions (Kerscher et al., 2006; Ikeda and Dikic, 2008). Multiple topologies of
polyubiquitin chains with different lysine linkage have been found (Ikeda and Dikic,
2008). It is known that the Lys48-linked polyubiquitin chains target substrates to the
proteasomal degradation, while the Lys63-linked polyubiquitin chains label
membrane proteins for endocytosis (Ikeda and Dikic, 2008). Although quite few,
some studies have also reported that cystein residues or the N-terminus of substrate
proteins are also possible sites for ubiquitylation (Ciechanover and Ben-Saadon, 2004;
Cadwell and Coscoy, 2005).
The 26S proteasome is a complex of ATP-dependent proteases that is capable of
recognition and degradation of ubiquitylated proteins in eukaryotes (Coux et al., 1996;
Baumeister et al., 1998). The 26S proteasome consists of two parts, the regulatory
19S particle and the hydrolytic 20S proteasome. The 20S proteasome is formed with
two inner β-rings assembled as a proteolytic chamber and one outer α-ring on each
side, which serves as the gate for the entry of substrates into the proteolytic chamber.
The 19S regulatory particle could also be divided into two subcomplexes, the base
and the lid. While the base subcomplex harbors the ATPase activity and the
ubiquitin-binding domain, which is response for the denaturing of substrates as well
as the opening of α-rings, function of the lid subcomplex is not fully known yet,
which is only shows to have de-ubiquitylation activity so far (Murata et al., 2009).
As earlier mentioned, proteins that are modified with Lys48 polyubiquitin chains
will be degraded by proteasomes. The Lys48 polyubiquitin chains are first recognized
by the 19S regulatory particle, which also denatures substrates and opens the α-rings
of the 20S proteasome by the activity of the ATPase. After removing the
poly-ubiquitin chains from substrates by the lid, substrates are sent into the proteolytic
chamber of the 20S proteasome for degradation and releasing free amino acids for
reuse (Murata et al., 2009).
Overview of autophagy
Autophagy, a conserved process for the degradation of long-lived proteins and
organelles in eukaryotes, is a major pathway for protein turnover in response to
environmental stresses (Tsukada and Ohsumi, 1993; Levine and Klionsky, 2004). The
operation of autophagy could be dissected to several stages. First is the induction of
autophagy, which is triggered by the deprivation of nitrogen source in yeast(Levine
and Klionsky, 2004); under nutritional condition, Atg1 and Atg13 are
hyper-phosphorylated through the action of the mTOR complex and dissociated from
each other, leading to the conduction of a pathway, called the cytoplasm-to-vacuole
targeting (Cvt) pathway, which is similar to autophagy but with a much lower
transport capacity (Jung et al., 2010; Kamada et al., 2010). The Cvt pathway, which is
now regarded as a specific type of selective autopahgy (Klionsky et al., 1992; Scott et
al., 1997; Lynch-Day and Klionsky, 2010), is activated constitutively under nutritional
condition for delivery of some vacuolar enzymes. Precursors of the vacuolar
aminopeptidase Ⅰ (prApe1) are synthesized in cytosol and self-oligomerize into an
electron dense structure, the Cvt complex. In addition to prApe1, the Cvt complex at
least contains another vacuolar enzymes, α-mannosidase. Atg19, the specific receptor
for prApe1 and α-mannosidase transport, mediates the interaction between the Cvt
complex and Atg11, which triggers the formation of Cvt vesicles leading to the
transport of prApe1 and α-mannosidase to the vacuole. While as under nutrition
limitation conditions, the activity of mTOR is inhibited, leading to the partial
de-phosphorylation and subsequent association of Atg1, Atg13, and their associated
proteins to form multi-protein complex, which stimulates the progression of
autophagy (Jung et al., 2010; Kamada et al., 2010).
Once autophagy is activated, a double membrane sac structure termed isolation
membrane starts to assemble, elongate, and expand from a specific cytosolic location,
the pre-autophagosomal structure or the phagophore assembly site (PAS). The edge of
the membrane structure eventually fuses to form a double membrane vesicle, called
autophagosome, which sequesters a portion of cytoplasm including cytosolic proteins
and organelles. The elongation of the isolation membrane into autophagosomes
depends on two conjugation systems made of two ubiquitin-like proteins, Atg8 and
Atg12 (Ohsumi, 2001). Atg12 is activated by the E1 like enzymes Atg7, subsequently
transferred to the E2 like enzyme Atg10, and finally irreversibly conjugated to Atg5.
Atg12-Atg5 conjugate binds with Atg16 to form a ~350 kD multimeric protein
complex. Atg8, which is synthesized as a precursor form, is processed by Atg4
protease, sequentially interacted with Atg7 andATg13, and eventually conjugated to
phosphatidylethanolamine (PE). It was proposed that the Atg12-Atg5-Atg16 complex
acts as the E3 enzyme for the Atg8 conjugation system (Hanada et al., 2007). The role
of the Atg8-PE conjugate in autophagy is not clear yet, but it was proposed to be
involved in the regulation of membrane tethering and hemifusion (Nakatogawa et al.,
2007). Other studies also show that Atg8 controls the expansion of isolation
membrane and determines the size of autophagosomes (Xie et al., 2008).
The membrane source of autophagosomes is an issue still under debate. PAS, the
structure regarded as the site of vesicle formation during autophagy (Suzuki et al.,
2001; Kim et al., 2002), has high concentration of phosphatidylinositol 3-phosphate
(PI3P) required for autophagy progress that is synthesized in site by the
phosphatidylinositol (PtdIns) 3-kinase complex I, which includes the PtdIns-3-kinase
Vps34 and its regulatory components such as Vps15, Vps30/Atg6, and Atg14 (Kihara
et al., 2001). The transmembrane protein Atg9, which cycles between the PAS and
peripheral structures, appears to regulate vesicle formation likely by, providing lipid
components for the expansion of isolation membrane. Recently, some Rab GTPase
and their guanine-nucleotide-exchange factors (GEFs) involved in the functions of the
Golgi complex or traffic from ER to the Golgi complex are shown to contribute to the
vesicle formation step during autophagy (Reggiori et al., 2004; Geng et al., 2010;
Lynch-Day et al., 2010; van der Vaart et al., 2010; Yen et al., 2010). Moreover,
mitochondria were shown to be one membrane source during autopahgy in
mammalian cells (Hailey et al., 2010). However, it is worth to mention that whether
defect in autophagy is due to the block in specific transport steps that directly
contributes to autophagosome formation or the impairment of membrane flow in the
early secretory pathway that indirectly affects autophagy is not always easy to
distinguish..
Autophagosomes sequester cytosolic proteins or organelles and transport cargoes
from cytosol to vacuoles for degradation (Levine and Klionsky, 2004; Suzuki and
Ohsumi, 2007; Xie et al., 2008). In mammalian cells, autophagosomes were shown to
move in a microtubule-dependent way (Monastyrska et al., 2009). One of the
homologues of Atg8 in mammalian cells termed microtubule-associated protein 1
light chain 3, MAP1-LC3 or simply LC3 in short, anchors autophagosomes to dynein,
which carries autophagosomes along the microtubule from plus end to minus end. In
addition, LC3 could associate with microtubule directly or indirectly via interaction
with MAP1A and MAP1B, increasing the affinity between microtubule and
autophagosomes that facilitates autophagosomes trafficking. Interestingly, although
Atg8 is homologous to LC3 (28% identity to rat MAP1-LC3) (Lang et al., 1998;
Reggiori and Klionsky, 2002), microtubules are not required for bulk autophagy in
yeast. Instead, actin filaments were shown to be required for the Atg11-dependent
selective types of autopahgy, but not nonspecific autophagy in yeast (Reggiori et al.,
2005).
Autophagosomes in the end fuse with the vacuole for degradation of cargoes. To
target autophagosomes properly to the vacuole in yeast, the required molecular
machinery includes the SNARE proteins Vam3, Vam7, Vti1, and Ykt6, the NSF,
SNAP, and GDI homologs Sec17, Sec18, and Sec19, the Rab protein Ypt7, and
members of the class C Vps/HOPS complex (Wang and Klionsky, 2003). After
vacuole docking, the outer membrane of autophagosomes fuses with the vacuolar
membrane, releasing inner vesicles, termed autophagic bodies, into the vacuolar
lumen. The limlting membrane of autophagic bodies would be lysed by the vacuolar
hydrolase Pep4, a process depends on the acidic environment of vacuolar lumen,
releasing the transported cytoplasmic components for degradation (Kim et al., 2001).
Finally, the efflux of amino acids resulting from autophagic degradation to the cytosol
is mediated by Atg22, Avt3, and Avt4, which are partially redundant vacuolar
effluxers to recycle amino acids. The recycled amino acids help maintain energy
homeostasis and protein synthesis under starvation (Yang et al., 2006).
The selectivity of autophagy
Starvation-induced autophagy was first regarded as a non-selective process. However,
increasing evidences have shown that cargo-recognition exists in the autophagic
sequestration stages, in which some proteins and orga12nelles are shown to be
degraded by autophagic process in a selective way (Kraft et al., 2009b). For example,
the Cvt pathway that is activated constitutively under nutrient rich condition is a
selective pathway in which Atg19 not only recognizes the self-oligomerized prApe1
as cargo but also interacts with Atg11 to stimulate the sequestration of prApe1 into
autophagosomes for delivery to the vacuole (Chang and Huang, 2007). Ribophagy,
the selective degradation of ribosomes under starvation, is a process unique in the
dependence of the ubiquitin protease Ubp3/Bre5 (Kraft et al., 2009a). Mitophagy, the
process autophagy that cells degrade excessive mitochondria under starvation, are
shown to be a selective autophagy that depends on the interaction between Atg11 and
a mitochondrial outer-membrane protein, Atg32 (Kanki and Klionsky, 2008; Kanki et
al., 2009; Okamoto et al., 2009). Pexophagy, the degradation pathway for the turnover
of excessive peroxisomes selectively under starvation, was shown to be an
Atg11-dependent pathway, too (Bellu and Kiel, 2003; Sakai et al., 2006). Cytosolic
proteins are also shown to be degraded selectively by autophagy in mammalian cells,
a process which is known to be mediated by ubiquitin-modification (Kirkin et al.,
2009b). Protein misfolding, which accompanies normal protein synthesis, could be
exacerbated by environmental stresses, such as oxidative stresses, starvation, and heat
shock (Goldberg, 2003). Misfolded proteins are recognized by chaperones, promoting
protein refolding or stimulating conjugation by ubiquitin for proteasomal degradation
(Imai et al., 2002; Kirkin et al., 2009b). These proteins, if left unresolved by
chaperone-dependent refolding or proteasomal degradation, will hamper cellular
metabolism, leading to death of the cell (Kirkin et al., 2009b). In response to the
toxicity of misfolded proteins, it is reported that misfolded proteins from aggregates
in a SQSTM1/p62 and NBR1 dependent process (Komatsu et al., 2007; Seibenhener
et al., 2007; Kim et al., 2008; Kirkin et al., 2009a). During the process, misfolded
proteins first are ubiquitylated, then the whole conjugate interacts with the UBA
domain of SQSTM1/p62 and NBR1. The PB1 domain of SQSTM1/p62 and NBR1
mediates self-oligomerization, which facilitates the formation of aggregates of
ubiquitylated misfolded proteins in the cytoplasm (Komatsu et al., 2007; Seibenhener
et al., 2007; Kim et al., 2008; Kirkin et al., 2009a; Kirkin et al., 2009b). While they
are inaccessible to be cleaned by the 26S proteasomes, protein aggregates are
selective substrates for autophagic degradation in mammalian cells (Kirkin et al.,
2009b). It is reported that the LIR motif of the p62/SQSTM1 and NBR1, which
interacts with LC3, mediates protein aggregates to be recognized by the autophagy
machinery, leading to the sequestration of protein aggregates into autophagosomes
and their transportation into lysosomes for degradation (Pankiv et al., 2007;
Seibenhener et al., 2007).
As a consequence, ubiquitylation not only labels proteins for the UPS degradation but
also marks cargo to be cleaned by autophagy in mammalian cells. Considering the
evolution-conserved roles of ubiquitins and autophagy, we tried to verify the function
of ubiquitin in autophagic degradation of cytosolic protein in yeast. To our surprise,
we discovered that ubiquitylation hinder, rather than promote the autophagic
degradation of cytosolic protein in yeast, which is opposite to that in mammalian cells.
Moreover, we identified an ubiquitin mutant that not only prevents the degradation of
ubiquitylated proteins by the 26S proteasomes but also eliminates the inhibitory effect
of ubiquitylation on autophagic degradation. We proposed that ubiquitylated proteins
might interact with an unknown ubiquitin-interacting protein in the cytosol that
hinders the sequestration of cytosolic proteins into autophagosomes for transportation
into vacuole and degradation. Our data clearly showed that ubiquitylation plays
different roles in autophagic degradation of cytosolic proteins in yeast versus
mammalian cells.
Materials and Methods Strains and Media
The yeast Saccharomyces cerevisiae starins used in this study are liested in Table 1.
Media used for growing yeast were listed below. SMD, 0.67% yeast nitrogen base
without amino acids, 2% glucose, 0.5% casamino acid, supplemented with
appropriate amino acids and vitamins; SD-N, 0.17% yeast nitrogen base without
amino acids and ammonium sulfate, 2% glucose, supplemented with appropriate
vitamins. Synthetic medium for yeast to grow overnight for proteasome inhibition
were modified from a published report (Liu et al., 2007), which consists of 0.17%
yeast nitrogenous base without amino acids and ammonium sulfate, 0.1% proline, 2%
glucose, supplemented with appropriate amino acids and vitamins.
Plasmid construction
Plasmids used in this study are listed in Table 2.
To express free EGFP in yeast, the pRS414 (or pRS416)-PCu-EGFP-TCYC1 plasmid
with the CUP1 gene promoter was constructed. Sequence encoding EGFP ORF was
amplified by PCR. Two restriction enzymes sites, HindⅢ and ClaⅠ, were introduced
to the ends of the PCR products. The resulting EGFP coding sequence was cloned into
the same sites of the pRS414-PCu-TCYC1 vector.
To express UbG76V-EGFPF in yeast, the pRS414 (or pRS416)-PCu-UbG76V-
EGFP-TCYC1 plasmid with the CUP1 gene promoter was constructed. Sequence
encoding ubiquitin ORF with the 76th glycine residue replaced with valine was
amplified by PCR. Two restriction enzyme sites, SpeⅠ and XmaⅠ, were introduced
to the ends of the PCR products. The resulting ubiquitin coding sequence was cloned
into the same sites of the pRS416-c-EGFP-TCYC1 vector (previously constructed in
our lab). Subsequently, the coding sequence of UbG76V-EGFP was amplified by PCR
with the introduction of a SpeⅠ and a ClaⅠ restriction sites to the ends of the
products. The PCR products were then subcloned into the same restriction sites of the
pRS414 (or pRS416)-PCu-TCYC1 vector.
To express UbI44A,G76V-EGFPF in yeast, the pRS414 (or pRS416) -PCu-UbI44A,G76V-
EGFP-TCYC1 plasmid with the CUP1 gene promoter was constructed. Sequence
encoding ubiquitin ORF with the 44th isoleucine and the 76th glycine residues replaced
with alanine and valine, respectively, was amplified by PCR. Two restriction enzyme
sites, SpeⅠ and XmaⅠ, were introduced to the ends of the PCR products. The
resulting ubiquitin coding sequence was cloned into the same sites of the
pRS416-c-EGFP-TCYC1 vector (previously constructed in our lab). Subsequently, the
coding sequence of UbI44A,G76V-EGFP was amplified by PCR with the introduction of
a SpeⅠ and a ClaⅠ restriction sites to the ends of the sequence. The PCR products
were then subcloned into the same restriction sites of the pRS414 (or
pRS416)-PCu-TCYC1 vector.
To express Pgk1-EGFP in yeast, the pRS416-PCuPgk1-EGFP-TCYC1 plasmid with
the CUP1 gene promoter was constructed. Sequence encoding PGK1 ORF was
amplified by PCR. Two restriction sites, BamHⅠ and HindⅢ, were introduced to the
ends of the PCR products. The resulting PGK1 coding sequence was cloned into the
same sites of the pRS416-PCu-EGFP-TCYC1 plasmid.
Western blot analysis
Culture aliquots harvested for western blot analysis were first precipitated by 10%
Trichloroacetic acid (TCA) for at least one hour and then washed with acetone three
times. After discard of the acetone, air-dried pellets were lysed in sample buffer
(1.25% SDS, 125mM Tris-Cl pH7.5, 12.5% glycerol, 0.0625% bromophenol blue and
1% β-mercaptoethanol) by vortex with silica beads (BioSpec Products, Inc.,
Bartlesville, OK) for 5 minutes and then boiled at 95℃ for another 5 minutes.
Centrifugation was performed to spin down cell debris. Samples were subjected to
SDS-PAGE, and then transferred to PVDF (polyvinylidene fluoride) membrane for
antiserum analysis. Bands of interested proteins were quantified and further analyzed
with Student’s T-test.
Fluorescent microscopy analysis
For the analysis of cells under nutrient rich condition, culture grown to mid-log phase
was harvested. Cells were washed by ddH2O once and then resuspended in ddH2O to
be smeared on slides for fluorescent microscopy analysis. For the analysis of cells
under starvation condition, cells of mid-log phase were transferred to SD-N for 4hr
cultivation. Cells were then harvested and treated as samples of nutrient rich condition
for fluorescent microscopy analysis.
Inhibition of proteasome activity with the treatment of MG132
Protocol for proteasome inhibition in yeast was modified from a published report (Liu
et al., 2007). Cells for proteasome inhibition were grown in synthetic medium
overnight, and then diluted into fresh synthetic medium to 0.1 OD600 for 10hr of
cultivation till concentration at OD600 = 0.6-0.8. 13.5 OD600 of cells were then
harvested and reintroduced into fresh synthetic medium containing 0.003% SDS for
3.5hr of cultivation. Next, 15 OD600 of cells were harvested and shifted into SD-N
containing 0.003% SDS and 75μM MG132 (Sigma-Aldrich, Inc.) for starvation.
For inhibition of protein synthesis and proteasome activity under starvation, cells
were first treated as previous described and then shifted to SD-N containing 0.003%
SDS, 75μM MG132, and 10μμg/ml Cycloheximide (Sigma-Aldrich, Inc.).
Results
Ubiquitylation triggers no protein aggregation in yeast
Ubiquitiylated red fluorescent protein (RFP) was used as a model to test the
degradation of ubiquitylated protein by autophagy in mammalian cell (Kim et al.,
2008). As the N-terminus of RFP is modified with a single ubiquitin molecule in
which the Gly76 was changed into Val (G76V) to prevent the cleavage of the ubiquitin
molecule from the ubiquitylated RFP by deubiquitinating enzymes (DUBs), it is
shown that the pattern of the red fluorescence signal turned from the diffusive
distribution to protein aggregates in cytosol by a p62 dependent pathway. Moreover,
protein aggregates showing signal of red fluorescence were highly-colocalized with
lysosomes, indicating that these protein aggregates were sequestered into
autophagosomes and subsequently degraded in lysosomes.
In order to clarify the role of ubiquitylation in the degradation of cytosolic protein
via autophagy in budding yeast Saccharomyces cerevisiae, we expressed ubiquitin
fusion degradation (UFD)-targeted EGFP, which was first reported as a powerful tool
for elucidation of the UPS system(Dantuma et al., 2000), for the analysis of protein
aggregation and their degradation via autophagy (Figure 1A). Two N-terminus
ubiquitylated EGFP were expressed. UbG76V-EGFP is a fusion protein that the EGFP
is modified with an ubiquitin moiety habors a G76V mutation to prevent the cleavage
of ubiquitin from the fusion protein. UbI44A,G76V-EGFP is another fusion protein, in
which the EGFP is modified with an ubiquitin moiety that the Ile44 was mutated to Ala
(I44A) in addition to the G76V mutation. From previous review, we know that Ile44 is
the key epitope in most interactions between ubiquitin-binding proteins and ubiquitin
(Chen and Sun, 2009), for example, p62, which promotes autophagic degradation of
ubiquitylated proteins in mammalin cells, and Rad23 and Dsk2, which transport
ubiquitylated proteins to proteasomal degradation. We replaced Ile44 with Ala to
prevent the interaction between ubiquitin and ubiquitin-binding proteins. Our data
showed that the I44A mutation blocked the proteasomal degradation prominently
(Figure 3), suggesting that the I44A mutation did destroy the interaction between
ubiquitin and ubiquitin-binding proteins effectively (see later paragraph for more
description).
We decided to analyze the distribution of fluorescent signal in cells by fluorescent
microscopy. We found that the distribution of free EGFP in WT cells under nutrient
rich condition dispersed in cytosol (Figure 1B), which was similar to that of the free
RFP in mammalian cells (Kim et al., 2008). The vacuole showed up clearly with the
absent of EGFP signal, showing that free EGFP was accumulated in cytosol instead of
transported to the vacuole, where the massive degradation of proteins and organelles
occurred.
To our surprise, the distribution of UbG76V-EGFP showed complete different
phenotype to that of the ubiquitylated RFP in mammalian cells. UbG76V-EGFP
dispersed throughout cytosol instead of forming protein aggregates (Figure 1B). The
vacuole was also absent of fluorescent signal, showing that the UbG76V-EGFP was not
the target of autophagy under nutrient rich condition. This result is different from that
of the ubiquitylated RFP in mammalian cells, which forms protein aggregates in
cytosol that is highly co-localized with lysosomes. The UbI44A,G76V-EGFP also
distributed diffusively throughout cytosol with the lack of fluorescent signal in the
vacuole (Figure 1B). UbG76V-EGFP, UbI44A,G76V-EGFP, and free EGFP were also
expressed in atg1Δ and pep4Δ cells as autophagy-defective strains. Atg1 is a
serine/threonine kinase required in the induction of autophagy and the formation of
autophagosomes as previously described (Jung et al., 2010; Kamada et al., 2010).
Pep4 is a vacuolar hydrolase charging for the breakdown of autophagic bodies
releasing into the lumen of the vacuole (Levine and Klionsky, 2004). Autophagy is
completely blocked in atg1Δ cells but remains its autophagosomes-transportation
activity in pep4Δ strain. The distribution of UbG76V-EGFP, UbI44A,G76V-EGFP, and free
EGFP expressed in atg1Δ and pep4Δ were similar to that of WT cells, indicating that
ubiquitylation triggers no protein aggregation and its autophagic degradation in yeast
under nutrient rich condition. However, it is possible that the strong signal of
diffusive fluorescent signal obscured the presence of protein aggregates in cytosol. We
decided to treat cells with 0.1% Triton X-100, leading to the leakage of soluble
cytosolic proteins from cytosol to outer environment. As a result, the structure of
protein aggregates, if any, could be seen clearly. Mid-log phase WT cells were first
treated with PBS solution containing 0.1% Triton X-100 for 30 minutes, then cells
were transfer into PBS solution containing 4% paraformaldehyde for 40 minutes for
fixation. Surprisingly, both three groups displayed similar fluorescent puncta in
cytosol. Although how these florescent puncta were formed in cytosol is not clear, no
difference could be observed between UbG76V-EGFP, UbI44A,G76V-EGFP, or free EGFP
expressing WT cells. From our data, We concluded that ubiquitylation triggers no
protein aggregation in yeast.
Ubiquitylated EGFP is a substrate for autophagic degradation
Non-selective autophagy is activated under starvation, in which cytosolic proteins are
sequestered into autophagosomes, and then delivered to the vacuole for degradation
(Levine and Klionsky, 2004). We decided to study the localization of ubiquitylated
proteins in cells under starvation by fluorescent microscopy analysis. Cells grown to
the mid-log phase were transferred to the nitrogen-starvation medium (SD-N) for 4hr
of starvation before observation. Our data showed that UbG76V-EGFP and
UbI44A,G76V-EGFP, were the target of starvation-induced autophagy in WT cells, which
were similar to that of free EGFP (Figure 2). Starvation stimulated the translocation of
UbG76V-EGFP and UbI44A,G76V-EGFP from cytosol to the vacuole in WT cells,
increasing the fluorescent signal in the vacuole. In atg1Δ cells which are lack of
autophagy activity, starvation stimulated no transportation of UbG76V-EGFP,
UbI44A,G76V-EGFP, and EGFP from cytosol to the vacuole (Figure 2). In pep4Δ cells
expressing UbG76V-EGFP, UbI44A,G76V-EGFP or free EGFP, fluoresecent signal
represented crowded autophagic bodies were detected clearly in the vacuole of pep4Δ
cells after 4hr of starvation (Figure 2). This is the direct evidence showing that
UbG76V-EGFP and UbI44A,G76V-EGFP were sequestered into autophagosomes as
cargoes to delivered to the vacuole under starvation.
Proteasomes, but not autophagy, degrade most ubiqutylated EGFP under
starvation
Our images indicated that UbG76V-EGFP and UbI44A,G76V-EGFP were transported to
the vacuole under starvation by an autophagy-depedent pathway, we decided to
analyze the different efficiency in their autophagic degradation by western blot
analysis. Since EGFP has a long half-life in the vacuole, ubiquitylated EGFP
transported to the vacuole was cleaved by hydrolases, releasing free EGFP in the
vacuolar lumen and can be detected with western blot by using anti-GFP antibody
easily. In comparison, ubiquitylated EGFP targeted to proteasomal degradation would
be digested into pieces, remaining no free EGFP. WT, atg1Δ and pep4Δ strains
expressing UbG76V-EGFP or UbI44A,G76V-EGFP were grown in SMD medium till the
mid-log phase, and then transferred into SD-N medium for starvation treatment.
Culture aliquots were collected at 0hr, 1hr, 2hr, 4hr and 6hr during a period of 6hr
starvation and precipitated with 10%TCA before western blot analysis. Using Pgk1 as
the loading control, the decrease of fusion proteins and the augmentation of free
EGFP allow us to monitor the difference between the degradation of UbG76V-EGFP
and UbI44A,G76V-EGFP under starvation.
UbG76V-EGFP decreased rapidly in WT strain as cells were shifted to the SD-N
medium (Figure 3A). At the 2hr of starvation, a band corresponding to the 25kDa of
free EGFP was observed and maintained in similar levels at the following time points
(Figure 3A). Interestingly, the degradation of UbI44A,G76V-EGFP in WT cells under
starvation showed different pattern to that of UbG76V-EGFP. UbI44A,G76V-EGFP was
maintained in similar levels under starvation. Free EGFP which could be detected
even at the very beginning of starvation was accumulated prominently (Figure 3A),
indicating that UbI44A,G76V-EGFP, which was blocked in proteasomal degradation,
became a good substrate for autophagic degradation. Surprisingly, the degradation of
UbG76V-EGFP in atg1Δ cells was even faster than that in WT cells with no generation
of free EGFP (Figure 3B), indicating that UbG76V-EGFP was degraded by proteasomes
effectively in atg1Δ cells. However, UbI44A,G76V-EGFP expressing in atg1Δ cells was
also maintained in similar levels under starvation accompanied no generation of free
EGFP. On the other hand, UbG76V-EGFP expressing in pep4Δ cells was degraded in a
much lower rate compared to that in WT and atg1Δ cells (Figure 3C), indicating that
UbG76V-EGFP which was sequestered into autophagic bodies were accumulated in the
vacuolar lumen of cells, keeping UbG76V-EGFP from the digestion of proteasomes. In
addition, UbI44A,G76V-EGFP was maintained in relatively high levels throughout
starvation (Figure 3C), demonstrating that UbI44A,G76V-EGFP, which was blocked in
proteasomal degradation, was accumulated in autophagic bodies.
Two conclusions could be drawn from our data. First, the activity of proteasomes
hinders the autophagic degradation ofUbG76V-EGFP. Second, I44A is the exact mutant
that destroys the interaction between ubiquitin and ubiquitin-interacting proteins in
proteasomal degradation.
Autophagic degradation of UbG76V-EGFP was slower than that of
UbI44A,G76V-EGFP
We decided to inhibit the activity of proteasomal degradation by treating cells with
MG132, short peptide aldehydes that block active sites of the 26S proteasome (Lee
and Goldberg, 1996). Unfortunately, due to the multiple drug-resistance of yeast, the
impermeability of the plasma membrane hampered the inhibition of MG132 treatment
in WT cells. Increased drug permeability, like mutant strain (erg6Δ, pdr5Δ), is
necessary for the treatment of MG132 (Liu et al., 2007). Fortunately, several methods
have been developed to inhibit proteasome activity in WT cells (Pannunzio et al.,
2004; Liu et al., 2007). To inhibit proteasome activity, cells were grown in specific
synthetic medium with proline as the nitrogen source for 10hr till the mid-log phase
and then shifted to fresh medium containing 0.003% SDS for additional 3.5hr
cultivation. For starvation, cells were transferred into SD-N medium containing
0.003% SDS and 75μM MG132 for 6hr of cultivation. Culture aliquots were collected
and treated as previous described for western blot analysis.
The level of the UbG76V-EGFP was accumulated dramatically in WT cells under
starvation, while the level of the UbI44A,G76V-EGFP was remained constantly in WT
cells under starvation (Figure 4A). Quantification of these bands showed that the level
of the UbG76V-EGFP was accumulated remarkably compared to that of
UbI44A,G76V-EGFP, which was significantly higher after 2hr of starvation (Figure 4A).
On the other hand, while the level of free EGFP from the cleavage of UbG76V-EGFP,
which could be detected after 2hr of starvation, was increased gradually over times,
free EGFP from the cleavage of UbI44A,G76V-EGFP was detected after 1hr of starvation
with a rapid accumulation rate (Figure 4A). This result suggested that ubiquitylated
EGFP was synthesized and degraded in WT cells at the same time under starvation,
thus higher level of UbG76V-EGFP indicated that UbG76V-EGFP was degraded in a
much slower rate than that of UbI44A,G76V-EGFP by autophagic degradation. Moreover,
the I44A mutation destroyed the delay of ubiquitylated EGFP in autophagic
degradation, suggesting the participation of an unknown ubiquitin-interacting factor in
this process.
In autophagy-defective strains, we found no difference between UbG76V-EGFP and
UbI44A,G76V-EGFP in autophagic degradation. In atg1Δ cells, the level of fusion
proteins remained constant under starvation with no difference between UbG76V-EGFP
and UbI44A,G76V-EGFP (Figure 4B). Moreover, no free form EGFP was detected,
showing that the transport of both UbG76V-EGFP and UbI44A,G76V-EGFP to the vacuole
was blocked in atg1Δ cells. pep4Δ cells showed similar result to that of atg1Δ cells
(Figure 4C), in which the levels of fusion proteins decreased slightly that nearly
remained constant under starvation with no prominent difference between
UbG76V-EGFP and UbI44A,G76V-EGFP. No free EGFP was detected either, indicating
that no ubiquitylated EGFP was digested by vacuolar hydrolases in pep4Δ cells. These
results suggested that the synthesis of new ubiquitylated EGFP was blocked in atg1Δ
and pep4Δ cells, probably by the shortage of the nutrient-supply in
autophagy-defective cells. Ubiquitylated EGFP remains stable under starvation,
suggesting that levels of ubiquitylated EGFP in WT cells were primarily due to the
equilibrium between autophagic degradation and protein synthesis.
Low level of ubiquitylated EGFP is not a substrate for autophagic degradation
To eliminate the effect of new-synthesized protein in our analysis of ubiquitylated
EGFP degradation under starvation, we decided to treat cells with Cycloheximide
(CHX), an inhibitor of protein synthesis in eukaryotes produced by the bacterium
Streptomyces griseus. Cells were treated and collected as previous described except
the treatment of starvation, in which 10 μg/ml of CHX was added into the SD-N
medium in addition to 0.003% SDS and 75μM MG132 for the inhibition of protein
synthesis and proteasome activity in cells under starvation. Astonishingly,
quantification from the western blot analysis showed that the level of UbG76V-EGFP
processed a gradual decrease over starvation in WT cells with no appearance of free
EGFP. In comparison, UbI44A,G76V-EGFP was maintained in similar levels over
starvation with the gradual increase of free EGFP, indicating that UbI44A,G76V-EGFP
was processed by autophagic degradation normally (Figure 5A). In atg1Δ cells
(Figure 5B), UbG76V-EGFP was decreased in a much higher rate, in which the level of
fusion protein remained less than half of the initial level at the end of the 6hr
starvation, than that of WT cells. No free EGFP was detected from the degradation of
UbG76V-EGFP in atg1Δ cells. The level of UbI44A,G76V-EGFP also decreased under
starvation, however, in a much lower rate than that of UbG76V-EGFP with no free
EGFP detected as well.
From these data, we proposed that the decrease of UbG76V-EGFP in WT cells under
the treatment of MG132 and Cycloheximide under starvation was due to proteasome
activity that was not completely blocked by treatment of MG132, while the remaining
UbG76V-EGFP was escaped from autophagic degradation by an unknown mechanism
which was destroyed by the I44A mutation. Moreover, our data suggested that 26S
proteasome might maintain a higher activity in atg1Δ cells than WT cells, which
degraded ubiquitylated EGFP in a higher rate in autophagic-defective cells than WT
cell.
Delay of ubiquitylated EGFP in autophagic degradation is not unique to specific
genetic background
To test whether the delay of ubiquitylated EGFP in autophagic degradation is a strain
specific pathway, we expressed UbG76V-EGFP and UbI44A,G76V-EGFP in BY4742
strain. Cells expressing UbG76V-EGFP or UbI44A,G76V-EGFP are grown to mid-log
phase and treated as previous described for the inhibition of proteasome activity. Data
collected from BY4742 strain showed similar result to that of the SEY6210 (WT)
strain (Figure 6), in which UbG76V-EGFP was accumulated in a higher rate than that of
UbI44A,G76V-EGFP under starvation. A delay in the detection of free EGFP from the
cleavage of UbG76V-EGFP compared to that of UbI44A,G76V-EGFP was similar to data
from SEY6210 strain. These data showed that the delay of ubiquitylated EGFP in
autophagic degradation is not a strain-specific situation.
Autophagic degradation of ubiquitylated EGFP is slower that of other cytosolic
proteins
Our data showed that autophagic degradation of UbG76V-EGFP was hampered
compared to that of UbI44A,G76V-EGFP. However, we could not rule out a possibility
that I44A mutation created a super substrate facilitating autophagic degradation of
ubiquitylated EGFP. To verify that I44A mutant is actually the mutation which
destroyed the delay of ubiquitylated EGFP in autophagic degradation, we analyzed
the difference between ubiquitiylated EGFP and other cytosolic proteins in autophagic
degradation under starvation..
We expressed a fusion protein Pgk1-EGFP in WT cells as an indicator for
non-selective autophagic degradation, in which Pgk1 is C terminally modified with a
EGFP that would be released after the cleavage of fusion protein by vacuolar
hydrolases in the vacuole. Pgk1 is a 3-phosphoglycerate kinase that catalyzes transfer
of high-energy phosphoryl groups from 1,3-bisphosphoglycerate to ADP to produce
ATP, which mainly located in cytoplasm of cells and was used as the internal control
in western blot analysis. Pgk1-EGFP expressed in yeast as a fusion protein was
delivered to the vacuole under starvation for hydrolases digestion into fragments,
releasing free EGFP accumulated in the vacuole. Thus the level of endogenous Pgk1
may not be affected by the cleavage of Pgk1-EGFP.
We first analyzed the distribution of Pgk1-EGFP in cells by fluorescent microscopy.
WT, atg1Δ, and pep4Δ cells expressing Pgk1-EGFP were grown in SMD till mid-log
phase as the sample of nutrient rich condition for fluorescent microscopy analysis.
Images of WT, atg1Δ and pep4Δ cells under nutrient rich condition showed similar
phenotype, in which Pgk1-EGFP dispersed throughout cytosol under nutrient rich
condition with the vacuole absent of fluorescent signal (Figure 7A). Next we
transferred mid-log phase cells into SD-N for 4hr of starvation and then analyzed the
distribution of Pgk1-EGFP in cells under starvation by fluorescent microscopy
(Figure 7A). Images of WT cells showed that the signal of Pgk1-EGFP was detected
in the vacuole, indicating that starvation triggered the transport of Pgk1-EGFP from
cytosol to the vacuole. Images from atg1Δ cells showed that starvation triggers no
transport of Pgk1-EGFP from cytosol to the vacuole, indicating that autophagy played
critical role in the transport of Pgk1-EGFP from cytosol to the vacuole in WT cell.
Moreover, accumulated autophagic bodies labeled with fluorescent signal were
detected in the vacuole of pep4Δ cells, confirming that Pgk1-EGFP was sequestered
into autophagosomes for autophagic degradation.
The level of Pgk1-EGFP degradation was further determined by western blot
analysis. Cells expressing Pgk1-EGFP were treated as previous described, in which
cells were cultivated in SD-N medium containing 75μM MG132 for a period of 6hr
starvation. The level of Pgk1-EGFP in WT cells remained constant over starvation
followed with an increase of free EGFP which was first detected after 1hr of
starvation (Figure 7B). In atg1Δ and pep4Δ cells, PgK1-EGFP was kept in a constant
level with no free EGFP was generated (Figure 7B). Quantification of these bands
showed that Pgk1-EGFP expressing in WT cells was maintained in similar level with
UbI44A,G76V-EGFP expressing in WT cells (Figure 7C). The accumulation of free
EGFP from the cleavage of Pgk1-EGFP also displayed a similar pattern to that of
UbI44A,G76V-EGFP in WT cells (Figure 7C). However, WT cells significantly
accumulated more UbG76V-EGFP than Pgk1-EGFP under starvation, showing that
Pgk1-EGFP was more readily to be degraded by autophagy in WT cells (Figure 7C).
In atg1Δ cells and pep4Δ cells, Pgk1-EGFP, UbG76V-EGFP, and UbI44A,G76V-EGFP
were maintained in similar levels under starvation (Figure 7D, 7E).
Based on these data, we clearly demonstrated that compared to other cytosolic
proteins, UbG76V-EGFP was delayed in autophagic degradation.
Discussion
Ubiquitylation triggers no protein aggregation in yeast.
Our data suggested that ubiquitylation stimulates no protein aggregation in budding
yeast. It is known that SQSTM1/p62 and NBR1 trigger ubiquitylated protein
aggregation in mammalian cells, which are further selectively degraded by autophagy
(Kim et al., 2008; Kirkin et al., 2009a). However, here we showed that signal of
UbG76V-EGFP and UbI44A,G76V-EGFP dispersed throughout cytosol in yeast, similar to
that of free EGFP (Figure 1). This suggested that ubiquitylation plays no role in
protein aggregation in yeast. Although some fluorescent punta which might represent
protein aggregates were observed after treating cells with 0.1% triton X-100 and 4%
paraformaldehyde, no difference could be detected between cells expressing
UbG76V-EGFP, UbI44A,G76V-EGFP, or free EGFP. This indicated that the formation of
these fluorescent puncta is not an ubiquitin-dependent pathway. From these data, we
hypothesize that no SQSTM1/p62 or NBR1 analogs present in yeast.
Delay, rather than acceleration, of UbG76V-EGFP in autophagic degradation
Ubiquitylation triggers not only proteasomal degradation but also lysosomal
degradation of proteins in mammalian cells. This has been regarded as a protective
pathway for cell survival under environmental stresses. However, this mechanism
seems not to exist in yeast. Moreover, we showed that ubiquitylation partially hinders
EGFP from autophagic degradation (Figure 4). This result supports our hypothesis
that no SQSTM1/p62 or NBR1 analogs are present in yeast. First, from images of
fluorescent microscopy (Figure 2), we showed that UbG76V-EGFP, UbI44A,G76V-EGFP,
and free EGFP would be translocated from cytosol to the vacuole in response to
starvation in an autophagy-dependent pathway, which is also supported by the
emergence of free EGFP in WT cells expressing UbG76V-EGFP or Ub I44A,G76V-EGFP
in western blot analysis (Figure 3). However, after75μM MG132 was used to block
proteasome activity under starvation, UbG76V-EGFP started to accumulate in WT cells,
which was significantly higher than the level of UbI44A,G76V-EGFP after 1hr of
starvation. Moreover, free EGFP cleaved from UbI44A,G76V-EGFP in WT cells was
accumulated in a higher rate than that from UbG76V-EGFP (Figure 4A). Compared to
the degradation rate of Pgk1-EGFP, we demonstrated that UbG76V-EGFP, but not
UbI44A,G76V-EGFP, is delayed in autophagic degradation (Figure 7). This is the direct
evidence showing that ubiquityltion, which targets protein to proteasomal degradation,
delays autophagic degradation of EGFP, indicating that ubiquitylated proteins could
somehow escape from the non-selective sequestration of cytoplasm into
autophagosomes for vacuolar degradation by an unknown pathway.
Low level of ubiquitylated EGFP is not a substrate for autophagy
To eliminate the effect of new-synthesized protein in our analysis of ubiquitylated
EGFP degradation under starvation, we decided to treat cells with CHX and MG132
to inhibit protein synthesis and proteasome activity under starvation. Surprsingly,
UbG76V-EGFP was degraded gradually in WT cells under starvation accompanied no
free EGFP (Figure 6A). However, free EGFP was still detected in UbI44A,G76V-EGFP
expressing cells (Figure 6A), indicating that low level of UbG76V-EGFP was targeted
to proteasomal degradation rather than autophagy. Combined with previous results,
we concluded that low amount of UbG76V-EGFP would be destined to proteasomal
degradation instead of delivering to vacuole under starvation, whereas high amount of
UbG76V-EGFP that exceeds the threshold would be sequestered into autophagosomes
for autophagic degradation.
I44A mutation destroys the delay of ubiquitylated EGFP in autophagic
degradation
We hypothesized that an unknown ubiquitin-interacting factor which binds to
UbG76V-EGFP prevents its autophagic degradation. This hypothesis is supported by
our data that an additional I44A mutation on ubiquitin moiety, which was shown to
block proteasomal degradation of UbI44A,G76V-EGFP effectively (Figure 3A), also
destroyed the delay of ubiquityled EGFP in autophagic degradation. This indicated
that the release of UbI44A,G76V-EGFP from the unknown ubiquitin-interacting factor
allows the sequestration of the fusion protein into autophagosomes for degradation.
Moreover, limited amount of the ubiquitin-interacting factor determines the threshold
that low amount of UbG76V-EGFP would be recognized and kept away from isolation
membrane whereas exceeded UbG76V-EGFP would be sequestered into
autophagosomes and delivered to the vacuole under starvation. On the one hand, this
unknown ubiquitin-interacting factor probably mediates proteasomal degradation of
UbG76V-EGFP. Dual roles might be played by this unknown ubiquitin-interacting
factor in degradation of ubiquitylated proteins.
The competition between proteasome and autophagy for UbG76V-EGFP as a
substrate
Our data demonstrated that UbG76V-EGFP was a substrate preferred to be degraded by
proteasomes rather than autophagy (Figure 3, 5). Under nutritional stage, new
synthesized UbG76V-EGFP is soon degraded by proteasomes, maintaining this protein
in a low level with no free EGFP generated. After cells are shifted to SD-N medium,
although UbG76V-EGFP is still targeted to proteasomes, the activation of autophagy
triggers the sequestration of cytoplasm into autophagosomes for vacuolar degradation,
leading to some degradation of UbG76V-EGFP via autophagy, releasing free EGFP
(Figure 3A). This is supported from images of fluorescent microscopy that showed
the translocation of fluorescent signal from cytosol to the vacuole in WT cells under
starvation but not in atg1Δ cells (Figure 2). Paradoxically, our data also showed that
the decrease of UbG76V-EGFP was similar in WT and atg1Δ cells with proteasome
activity (Figure 3A, B), suggesting that first, autophagy may not contribute much to
the degradation of UbG76V-EGFP under starvation, and second, proteasome activity
might be up-regulated in atg1Δ cells.
It is interesting to point out that fusion proteins, especially UbG76V-EGFP, were
degraded in a higher rate in atg1Δ than WT cells when CHX and MG132 were used
to treat cells under starvation (Figure 5A, B). This supported our hypothesisi that
proteasome activity is up-regulated in autophagy-defective cells. Moreover, no free
EGFP was detected in WT cells (Figure 5A), suggesting that low level of
UbG76V-EGFP would be targeted to proteasomal degradation rather than autophagy.
This could be explained by our model that an unknown ubiquitin-interacting factor
mediates proteasomal degradation of ubiquitylated proteins and delays their
elimination by autophagy under starvation. It is now clear that multiple steps are
required in protein degradation through the UPS system (Funakoshi et al., 2002;
Richly et al., 2005; Ye, 2006; Dantuma et al., 2009). The substrate protein, by the
activity of E1, E2 and E3 enzymes, is first modified by one or two ubiquitin moieties.
This oligoubiquitylated substrate is then interacted with the Cdc48Ufd1/Npl4 complex
which further recruits Ufd2 as an “E4” enzyme extending the oligoubiquitin chain by
a few extra ubiquitin moieties. Subsequently, Rad23 (or Ddi1, Dsk2) is recruited to
the complex by the activity of Ufd2, which binds to the ubiquitylated substrate and
delivers it to the Rpn10, one component of the 19S proteasomal cap, for further
degradation. We propose that the unknown ubiquitin-interacting factor is one of these
proteins mediating the recognition of ubiquitylated protein by proteasomes. Cdc28,
Rad23 and Ddi1 were shown to interact with one or two ubiquitin-modified substrate,
while Dsk2 was shown to bind to polyubiquitin chain, preferentially to Lys28 linked
chain, via their UBA domain (Bertolaet et al., 2001; Funakoshi et al., 2002; Richly et
al., 2005; Dantuma et al., 2009). In addition, UbI44A,G76V-EGFP, which was poorly
interacted with the UPS machinery, was shown to be freely involved into autophagic
degradation. We believe that the interaction of ubiquitin moiety with these ubiquitin
binding factors led UbG76V-EGFP to proteasomal degradation and delayed their
sequestration into autophagosomes, probably by a resisting signal harboring in the
UPS system responding for the “cargo-recognition” process in starvation-induced
autophagy, which was originally thought to be a non-selective process.
It is interesting to note that Korolchuk et al. (2009) have reported that autophagy
inhibition compromises degradation of ubiquitin-proteasome pathway substrates in
mammalian cells, in which they showed that autophagy inhibition increases levels of
proteasome substrates. By the interaction of poly-ubiquitin chain with excessive p62,
ubiquitylated substrates which destined for proteasomal degradation originally
become protein aggregates accumulated in cytosol. (Korolchuk et al., 2009). This is
far different from our result, in which we proposed that proteasome activity is
up-regulated in autophagy-deficient cells, and ubiquitylated proteins targeted to
proteasomes are resistant to autophagic degradation. Combined with our fluorescent
images (Figure 1), we suggested that this is the another evidence showing that
mammalian p62 and NBR1 analogs are absent in budding yeast, leading to the fact
that proteasome machinery, but not autophagy, is the pathway that eliminates
ubiquitylated proteins in yeast. Autophagy are regarded as homologous pathways
among eukaryotes that share similar machinery, while our study suggested that the
role of ubiquitylation in autophagic degradation of soluble proteins is quite different
from mammalian cell to budding yeast.
The first report that ubiquitylation hinders autophagic degradation of cytosolic
soluble proteins
UPS system and autophagy are two parallel routes for protein degradation in
mammalian cells, in which autopahgy was regarded as a protective pathway cleaning
up misfolded proteins that are failed to be eliminated by proteasomes. It is known that
the impairment of constitutive autophagy causes cytoplasmic accumulation of
ubiquitylated inclusion bodies accompanied with severe liver injuries and
neurodegeneration.in mammals (Komatsu et al., 2007). However, here we showed
that ubiquitylated proteins are mainly degraded by proteaomes and was hindered from
autophagic degradation in budding yeast. A great puzzle was raised, how can yeast
deal with the accumulation of misfolded proteins under stresses without massive
degradation of ubiquitylation-dependent autophagy? We have shown that no
mammalian p62 or NBR1 analogs are presented in yeast to compete ubiquitylated
proteins with proteasomal machinery. However, it is not clear whether p62 and NBR1
are evolved independently in mammals or lost in budding yeast. Moreover, we could
not rule out a possibility that there exist a complementary pathway which is
independent of ubiquitylation in regulating level of misfolded proteins by autopahgy
in yeast. Thus, roles of autophagy in the clearance of misfolded proteins under
stresses must be studied. Budding yeast has been used to model neurodegeneration
elucidating mechanism underlying these complex diseases and developing novel
therapeutics (Khurana and Lindquist, 2010). However, our study has suggested
different roles of ubiquitylation in autophagic degradation of soluble protein between
yeast and mammalian cells, which should now be taken into consideration in working
with yeast as a model of neurodegenerative diseases. Nevertheless, some proteins, for
example, PrPSC in Prion diseases, are self-aggregation with no ubiquitylation required.
Further analysis of how protein aggregates are degraded by autophagy in yeast would
help us understanding cell toxicity in humans.
Finally, this is the first report that ubiquitylation does not promote autophagic
degradation of cytosolic protein. The different scenario in autophagic degradation of
ubiquitylated protein between yeast and mammalian cells reminds us that the
conserved pathway between different model systems may display great difference in
detail.
