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INTRODUCTION |
Vesicular transport of proteins is mediated by
coated vesicles that bud from membrane-bound compartments and fuse with
a target acceptor organelle. This process is highly conserved from
yeast to man. Docking of a vesicle at the appropriate target
membrane involves an interaction between integral membrane proteins
located on the vesicle, the
v-SNAREs,1 and the t-SNAREs
at the target membrane (1-3). After fusion of the membranes, the
tightly bound v-t-SNARE complex binds two soluble factors:
N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (SNAP). NSF catalyzes SNARE complex
disassembly via its ATPase activity, thus allowing a new round of fusion to take place (4-6).
GATE-16 (Golgi-associated ATPase
enhancer of 16 kDa) is an essential component of
intra-Golgi transport (7) and post-mitotic Golgi re-assembly (8).
GATE-16 is recruited to unpaired GOS-28, a Golgi-specific SNARE, in an
NSF/
-SNAP-dependent manner (7, 8), thus preventing
GOS-28 from interacting with its cognate t-SNARE syntaxin 5. As was
determined by its crystal structure (9), GATE-16 contains a ubiquitin
fold decorated by two additional NH2-terminal helices.
GATE-16 belongs to the ubiquitin-like protein family and shares
a high level of sequence identity with an expanding family of proteins
that have been implicated in a variety of cellular processes. Members
of this family include light chain 3, a subunit of the neuronal
microtubule-associated protein complex (10) recently implicated in
autophagocytosis (11, 12) and the GABA receptor-associated protein
(GABARAP), which was implicated in GABAA receptor
trafficking and post-synaptic localization (13-15). In
Saccharomyces cerevisiae there is only one known homologue of GATE-16, Aut7/Apg8, which was found to function in membrane dynamics
during autophagy (16, 17). This process constitutes a major pathway for
delivery of proteins and organelles to the lysosome or vacuole, where
they are degraded and recycled. Autophagy is particularly important
during development as well as under certain environmental stress
conditions such as nitrogen starvation. Aut7p is essential for
autophagosome biogenesis, playing a key role in the formation (12) and
elongation of autophagosomes (18). Despite its hydrophilic nature,
considerable amounts of Aut7p are covalently bound to membranes,
specifically conjugated to phosphatidylethanolamine (PE) (19, 20). For
this conjugation to occur, Aut7p must undergo a series of
modifications. First, newly synthesized Aut7p is cleaved downstream of
glycine 116 (out of 117 AA) by Apg4p/Aut2p, a cytosolic cysteine
protease. Next, Aut7p becomes conjugated to PE through the exposed
COOH-terminal glycine by a ubiquitination-like mechanism, involving
Apg7p as an E1-like enzyme and Apg3p as an E2-like enzyme (20).
Finally, conjugated Aut7p is cleaved again by Apg4p downstream of
glycine 116 and thereby released from the membrane (19).
We have previously found that GATE-16 isolated from bovine brain lacks
its phenylalanine residue at position 117 (7). We now describe the
isolation and characterization of HsApg4A as a human protease of
GATE-16. We show that GATE-16 is cleaved downstream of a conserved and
essential glycine 116, both in vivo and in vitro.
We then utilize a cell-free assay to show that HsApg4A is sufficient to
cleave GATE-16. These findings, together with the identification of the
human Apg7 (21) and Apg3 (22), indicate that GATE-16 undergoes a
similar modification to that of its yeast homologue, Aut7. The
isolation of HsApg4A provides a new tool for further investigation and
characterization of the role of GATE-16 in intracellular membrane trafficking.
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EXPERIMENTAL PROCEDURES |
Cloning of the Human Apg4 Homologue--
Based on the DNA
sequence of HsApg4A (GenBankTM accession number AB066214),
we cloned an open reading frame of the HsApg4A cDNA by PCR, using
an expressed sequence tag clone (number 309651 from ResGen, Invitrogen)
as a template. This cDNA fragment was sequenced and found to
correspond to the authentic HsApg4A sequence. The fragment was then
subcloned into a pQE-30 expression vector (Qiagen), and the obtained
construct was designated pQE-30HsApg4A. This construct encodes a
recombinant protein tagged by His6 at the NH2 terminus.
Plasmid Construction and Site-directed Mutagenesis of
GATE-16--
The open reading frame encoding GATE-16 was amplified by
PCR and subcloned into a pcDNA3 plasmid (Invitrogen), upstream to a
sequence encoding for a hemagglutinin (HA) tag, to produce a protein
tagged by HA at the COOH terminus. This construct, designated pcDNA3GATE-16-HA, was used for the in vivo studies. Two
mutant genes were constructed based on the WT construct: G116A, in
which glycine 116 was replaced with alanine, and F115A, in which
phenylalanine 115 was replaced with alanine. Mutagenesis was carried
out by the Pfu DNA polymerase (New England BioLabs)
with an oligonucleotide containing 5'-TTCGCCTTC-3'
and 5'-GCCGGCTTC-3', respectively (the nucleotides
that were changed to generate the mutations are underlined).
To express GATE-16-HA as a recombinant protein, the cDNA encoding
GATE-16-HA was amplified by PCR from pcDNA3GATE-16-HA and subcloned
into a pQE-30 bacterial expression vector (Qiagen). This construct
encodes a recombinant protein tagged by His6 at the
NH2 terminus and HA at the COOH terminus. Similarly, pQE-30 vectors containing the mutants G116A and F115A were constructed.
Expression and Purification of Recombinant Proteins--
The
pQE-30 plasmid containing GATE-16-HA was transformed into the
Escherichia coli M15 (pREP4) strain. Cells were grown to an
OD600 of 0.3-0.6 and induced with 1 mM
isopropyl-l-thio-
-D-galactopyranosidase for 3-4 h at
37 °C. Following centrifugation in a Sorvall SLA-3000 rotor at 6000 rpm for 15 min, the pellet was resuspended in a breaking buffer (10 mM Hepes, pH 7.4, 200 mM KCl, 10 mM
-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride
(PMSF), 2 µg/ml leupeptin, 2 µg/ml aprotonin, and 2 µM pepstatin A) and lysed by high pressure (8000 p.s.i.).
The lysates were cleared by 30-min centrifugation at 40,000 rpm in a
Beckman Ti60 rotor and then loaded onto a nickel beads column
(nickel-nitrilotriacetic acid (Ni2+-NTA), Qiagen). The
column was washed with washing buffer (10 mM Hepes, pH 7.4, 50 mM KCl, 10 mM
-mercaptoethanol)
containing 20 mM imidazole, and elution was carried out in
a gradient of 20-500 mM imidazole in the same buffer.
Samples were detected on SDS-PAGE, and the protein concentration was
determined by the Bradford assay (Bio-Rad). Fractions containing pure
GATE-16-HA were stored at
70 °C until use. A similar method was
used for the purification of recombinant His6-HsApg4A from
pQE-30HsApg4A, except for the omission of PMSF from the breaking buffer.
Expression of GATE-16 in Chinese Hamster Ovary (CHO)
Cells--
The pcDNA3GATE-16-HA plasmid was transfected into CHO
cells via electroporation (Bio-Rad kit) using 40 µg of DNA per 5 × 106 cells, according to the manufacturer's protocol.
Isolation of Cytosol from CHO Cells--
Cells were washed twice
with phosphate-buffered saline, then twice in a homogenization buffer
containing 0.25 M sucrose, 25 mM Tris-HCl, pH
7.4, and 50 mM KCl. The cells were then homogenized in the
same buffer containing 1 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml aprotonin, and 2 µM pepstatin A using a Balch
homogenizer in all experiments except the experiment described in the
legend to Fig. 1 in which a Dounce homogenizer was used. The
homogenates were centrifuged for 5 min at 2500 rpm, and the supernatant
was further centrifuged for 30 min at 200,000 × g. The
supernatant containing the cytosol was collected.
Isolation of Cytosol from Crude Rat Brains--
Brains were
homogenized in a breaking buffer (1 M KCl, 250 mM sucrose, 25 mM Tris, pH 7.4, 2 mM EGTA (in 100 mM Tris 7.4), 0.5 mM 1,10-phenanthroline, 1 mM DTT, 10 µg/ml
aprotinin, 0.5 µg/ml leupeptin, 2 µM pepstatin A, and 1 mM PMSF) with a Dounce homogenizer using a B handle. The
homogenate was then centrifuged in a Sorvall SS-34 rotor at 8500 rpm
for 1 h and subsequently cleared by 1.5-h centrifugation at 35,000 rpm in a Beckman Ti60 rotor. The supernatant was dialyzed for 3 × 2 h against 5 liters (total) of dialysis buffer (50 mM
KCl, 25 mM Tris, pH 7.4, and 10 mM
-mercaptoethanol), cleared again at 35,000 rpm for 1.5 h, and
stored at
70 °C until use.
Mass Spectrometry--
Protein samples were prepared from pure
untagged recombinant GATE-16 (9) by incubation with HsApg4A in the
presence of 1 mM DTT for 1 h. Mass spectra were
acquired on API Q-STAR Pulsari
electrospray-quadrupole time-of-flight mass spectrometer (MDS-Sciex and Applied Biosystems, Toronto, Canada) equipped with
nano-electrospray ion source for sample introduction.
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RESULTS |
GATE-16 Undergoes COOH-terminal Cleavage in
Vivo--
GATE-16, a ubiquitin-like protein, was implicated in
intra-Golgi transport and post-mitotic Golgi re-assembly. GATE-16
cDNA encodes for a 117-amino acid-long protein that is terminated
by a VAYSGENTFGF sequence. Amino acid sequencing of five different tryptic peptides, obtained from endogenous GATE-16 isolated from bovine
brain, revealed that all but one were terminated by lysine or arginine
residues. The peptide corresponding to the COOH terminus of the
protein, however, was found to be VAYSGENTFG (7). This finding alluded
to the possibility that cytosolic GATE-16 lacks its last amino acid as
a result of a specific protease activity. Consistently, it has been
recently reported that Apg4p, a novel cytosolic cysteine protease,
specifically removes a single amino acid from the COOH terminus of
Aut7p, the yeast homologue of GATE-16 (19).
To investigate whether GATE-16 undergoes COOH-terminal cleavage by as
yet an unknown protease, we expressed GATE-16 with a HA epitope
attached to its carboxyl terminus (GATE-16-HA) in CHO cells. Cells were
harvested after 48 h, and the cytosols were analyzed by Western
blot using anti-HA and anti-GATE-16 polyclonal antibodies. In addition,
two mutants in which glycine 116 or phenylalanine 115 were replaced
with alanine (G116A-HA and F115A-HA, respectively) were used to
determine whether these residues were essential for cleavage of the
COOH terminus. The cleavage of the HA tag from GATE-16-HA is displayed
in Fig. 1. Notably, the mutant G116A-HA remained intact, while F115A-HA was processed as the wild type protein,
indicating that the highly conserved glycine 116, but not phenylalanine
115, is essential for the cleavage of GATE-16.

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Fig. 1.
GATE-16 undergoes COOH-terminal cleavage
in vivo. CHO cells were transfected with the
mammalian expression vector pcDNA3 containing GATE-16
(WT) with an HA tag at its COOH terminus or mutants in which
glycine 116 or phenylalanine 115 of GATE-16 were replaced with alanine
(G116A and F115A, respectively). Cells were harvested 48 h after
transfection, and their cytosols (40 µg per lane) were analyzed by
Western blot using anti-HA and anti-GATE-16 antibodies as indicated.
The primary antibodies were reacted either by peroxidase-conjugated
anti-mouse or anti-rabbit, followed by enhanced chemiluminescence
detection.
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Characterization of the Cleavage Process--
A cell-free cleavage
assay was used to further characterize this COOH-terminal processing.
For that purpose, we produced recombinant GATE-16, tagged with six
histidine residues (His6), at the NH2 terminus
and HA at the COOH terminus. Similarly, a mutant protein, in which
glycine 116 was replaced by alanine, was produced. These proteins were
incubated at 30 °C with crude cytosols prepared from CHO cells or
rat brains for different durations of up to 1 h and subsequently
analyzed by Western blot, using anti-His monoclonal or anti-HA
polyclonal antibodies (Fig. 2).
Incubation of recombinant GATE-16-HA with CHO cytosol (or crude rat
brain cytosol, data not shown) for a period of 20-30 min (Fig.
2B) resulted in removal of the HA tag. The use of
anti-His6 antibodies (Fig. 2A, lower
panel) revealed that the protein was cleaved to a shorter form and
not degraded. In the control study, GATE-16-HA remained intact after
incubation for 1 h in the reaction buffer (25 mM Tris,
pH = 7.4, 50 mM KCl, and 10 mM
-mercaptoethanol) at 30 °C, in the absence of crude cytosol, or
in the presence of cytosol treated with the thiol alkylating agent NEM
(see below). These results demonstrate that after incubation with
cytosol, part of the COOH terminus of GATE-16 is cleaved. In accord
with the results shown in Fig. 1, we found that the conserved glycine
116 is essential for this cleavage process.

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Fig. 2.
Characterization of the cleavage process in a
cell-free system. GATE-16 (WT) and the mutant G116A
were cloned into pQE-30 vectors and expressed in E. coli to
produce recombinant proteins tagged with His6 at the
NH2 terminus and HA at the COOH terminus. Recombinant
His6-GATE-16-HA (1 µg) was incubated at 30 °C with CHO
cytosol (10 µg) in a reaction buffer that contained 25 mM
Tris, pH 7.4, 50 mM KCl, and 10 mM
-mercaptoethanol, under different conditions, and subjected to
immunoblotting with anti-HA polyclonal antibodies, unless otherwise
indicated. A, GATE-16 undergoes COOH-terminal cleavage by
CHO cytosol. His6-GATE-16-HA WT (lanes 2 and
3) or G116A (lanes 4 and 5) were
incubated with CHO cytosol for 1 h, in the presence (lanes
3 and 5) or in the absence (lanes 2 and
4) of 1 mM NEM. As a control,
His6-GATE-16-HA was incubated in a reaction buffer without
cytosol (lane 1). B, time course of the cleavage
reaction. His6-GATE-16-HA was incubated with CHO cytosol
for the indicated periods of time. As a control, incubation was carried
out for 1 h in the presence of 1 mM NEM. C,
the effect of protease inhibitors on the cleavage.
His6-GATE-16-HA was incubated with CHO cytosol in the
absence (lane 1) or in the presence of the following
reagents: 1 mM NEM (lane 2), 1 mM
PMSF (lane 3), 1 mM pepstatin A
(lane 4), 10 mM 1,10-phenanthroline (lane
5), 10 mM EGTA (lane 6), and 10 mM BAPTA (lane 7).
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We next tested the effect of different protease inhibitors on the
cleavage. As shown in Fig. 2C, the process was strongly inhibited by two factors: NEM, a thiol alkylating agent known to
inhibit cysteine proteases (lane 2), and PMSF, a
serine-protease inhibitor (lane 3). Addition of pepstatine
A, an aspartate-protease inhibitor (lane 4), or
1,10-phenanthroline, a metallo-protease inhibitor (lane 5),
did not affect the cleavage. Neither was the process affected by
Ca2+ chelating agents (EGTA and BAPTA, lanes 6 and 7, respectively). By contrast, addition of the reducing
agent DTT significantly increased the level of cleavage (data not
shown). These results could suggest that GATE-16 is processed by a
partially similar mechanism to that of Aut7p.
Identification of the Mammalian Homologue of Apg4/Aut2--
The
yeast Apg4p was recently suggested to have two putative homologues in
humans (Fig. 3A) (19). We
cloned HsApg4A and expressed it in E. coli, tagged with
His6 at the NH2 terminus. Following purification on a Ni2+-NTA-agarose columns, we detected
His6-HsApg4A as a major polypeptide of about 50 kDa (Fig.
3B).

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Fig. 3.
Identification of the human Apg4A as a
protease of GATE-16. A, alignment of the amino acid
sequences of the two proposed HsApg4s (HsApg4A, NCBI accession
number BAB83889, and HsApg4B, NCBI accession number BAB83890) with the
yeast pApg4/pAut2 (accession number NP_014176). The alignment was
preformed using the ClustalW multiple alignment program and is depicted
by the ESPript 2.0 program, representing identity by a black
frame and homology by a gray background. B,
recombinant HsApg4A is detected as a polypeptide of 50 kDa. The
cDNA of HsApg4A was cloned into a pQE-30 vector and expressed in
E. coli to create a His6-tagged recombinant
protein. Total bacterial cell lysate (lane 1) was loaded on
a Ni2+-NTA column and eluted by imidazole as explained
under "Experimental Procedures." The eluted fraction (lane
2) was run on 12% SDS-polyacrylamide gel and visualized by
Coomassie Brilliant Blue staining. C, HsApg4A cleaves
GATE-16-HA in vitro. Left panel, recombinant
His6-GATE-16-HA (1 µg) was incubated with recombinant
His6-HsApg4A (1 µg, lanes 1 and 2)
in a reaction buffer (see legend to Fig. 2), for 1 h, at 30 °C,
in the presence (lane 2) or absence (lane 1) of 1 mM DTT. As a control, GATE-16 was incubated with crude rat
brain cytosol (10 µg) in the presence (lane 4) or absence
(lane 3) of 1 mM NEM, or with a reaction buffer
only (lane 5), without cytosol or HsApg4A. Right
panel, the mutant His6-GATE-16-HA-G116A (1 µg) was
incubated with recombinant His6-HsApg4A (1 µg, lane
2), in the presence of 1 mM DTT (at the conditions
mentioned above). As a control, the mutant was incubated with a
reaction buffer only (lane 1). Proteins were subsequently
run on 15% SDS-PAGE and visualized by Coomassie Brilliant Blue
staining.
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To test whether HsApg4A is involved in the cleavage of GATE-16,
we incubated recombinant His6-GATE16-HA with recombinant
His6-HsApg4A, in the presence (Fig. 3C,
left panel, lane 2) or in the absence (lane
1) of 1 mM DTT (19). In the presence of DTT,
GATE-16-HA was cleaved to a shorter form; no cleavage was observed in
the absence of DTT. In addition, HsApg4A did not cleave the mutant form
of GATE-16, G116A (Fig. 3C, right panel,
lane 2). These results indicate that HsApg4A, in the
presence of the reducing agent DTT, is sufficient to cleave GATE-16 and
thus may function as the mammalian homologue of Apg4. To determine the
exact cleavage site of HsApg4A, we utilized pure recombinant GATE-16
isolated from E. coli with no tags attached to its
NH2 or COOH termini. Pure GATE-16 was incubated with
HsApg4A for 1 h at 30 °C, and the reaction mixture was
subjected to electrospray ionization mass spectrometry. While the
uncleaved GATE-16 yielded a single signal at 13,665.85, incubation with
HsApg4A produced an additional signal at position 13,518.89. These
signals correspond to the predicted molecular masses of the full-length
(13,666) and the processed (13,519) GATE-16, indicating that GATE-16
was cleaved by HsApg4A just after glycine 116.
To further characterize this enzymatic reaction, we incubated a
constant amount of GATE-16-HA with increasing amounts of HsApg4A (Fig.
4A) or crude rat brain cytosol
(Fig. 4B) for 1 h and then analyzed the proteins by
SDS-PAGE. Efficient cleavage of 1 µg (62 pmol) GATE-16 required at
least 0.5 µg (11 pmol) of HsApg4A. GATE-16 was not fully cleaved by
HsApg4A, even when larger amounts of HsApg4A were tested (up to 5 µg,
data not shown). Under the same conditions, cleavage of GATE-16 by
cytosol required 10 µg of rat brain cytosol. These results suggest
that either HsApg4 is an extremely abundant protein or more likely that
an additional cytosolic factor(s) is involved in the cleavage
process.

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Fig. 4.
Comparison of the cleavage activity of Apg4A
and crude cytosol. Recombinant His6-GATE-16-HA (1 µg) was incubated with increasing amounts of HsApg4A, 0.001-1 µg
(A), or crude rat-brain cytosol, 0.1-10 µg
(B), in a reaction buffer (see legend to Fig. 2) containing
1 mM DTT for 1 h at 30 °C. The proteins were
subsequently run on 15% SDS-PAGE, stained by Coomassie Brilliant Blue,
and quantified with a densitometer using the Bio-Rad multi-analyst
program. The black line represents the uncleaved GATE-16-HA,
whereas the gray line represents the cleaved form.
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DISCUSSION |
GATE-16 has been recently implicated in fusion processes
that take place in the Golgi apparatus (7, 8, 23). Although it lacks a
significant amino acid sequence similarity to ubiquitin, GATE-16 was
found to contain a single ubiquitin fold subdomain decorated by two
helices at its NH2 terminus (9). Like ubiquitin, GATE-16 is
synthesized as a precursor protein. GATE-16 has a single, COOH-terminal
phenylalanine residue following the conserved phenylalanine and glycine
residues, and it stands to reason that this terminal phenylalanine
residue must be removed for a conjugation process to take place. Here
we showed that HsApg4A, a specific cysteine protease, cleaves GATE-16
at its COOH terminus. We presented evidence that this cleavage takes
place in vivo and in vitro using either crude
cytosol or purified HsApg4A. Our results indicate that HsApg4A specifically removes a single amino acid from the COOH terminus of
GATE-16, thus exposing glycine 116. Furthermore, glycine 116 appears
essential for this process inasmuch as replacing it with alanine
abolishes the ability of GATE-16 to be cleaved. It appears that GATE-16
is a substrate for HsApg4A, which in turn renders it into a potential
substrate for a ubiquitin-like conjugase. In accord with this
suggestion, GATE-16, as well as its two other mammalian homologues,
GABARAP and LC-3, were recently shown to be covalently attached to
human homologues of E1 (Apg7)- and E2 (Apg3)-conjugating enzymes (Refs.
21 and 22, respectively).
HsApg4A exhibits significant amino acid sequence homology to the
yeast autophagic factor Apg4p, a novel cysteine protease that
specifically cleaves the carboxyl terminus of Aut7p downstream of
Gly-116 (19). Apg4p has been shown to cleave the newly synthesized Aut7p, a reaction that precedes a ubiquitination-like process, mediated
by E1 and E2 ligases, which specifically conjugates Aut7p to PE (20).
Apg4p then releases Aut7p to the cytosol by cleaving the Aut7p-PE bond.
These cleavage processes mediated by Apg4p are essential for both
autophagy and cytosol to vacuole (Cvt) pathways (19). Interestingly,
while in S. cerevisiae there is only a single Aut7p-like
protein and a single Apg4p enzyme, mammals possess at least 10 different GATE-16-like proteins and at least 2 Apg4-like proteases. A
possible explanation for this phenomenon is that HsApg4A cleaves the
newly synthesized GATE-16 as well as the other members of the family,
while its other mammalian homologue, HsApg4B, cleaves these proteins
from their targets (possibly PE). Alternatively, it is feasible that
different mammalian Apg4 proteins are specific to different subsets of
the GATE-16 family members. This may imply that the two Apg4s have a
different tissue specificity or subcellular localization or that they
may be expressed at different developmental stages. Indeed, it has been
recently reported that loss of one of the Apg4 homologues in
Drosophila melanogaster resulted in significant defects in cut and Notch signaling pathways (24).
HsApg4a activity is similar to that of the deubiquitination enzymes and
of SUMO-specific proteases. On the basis of their amino acid sequence
homology, deubiquitinating enzymes can be broadly divided into two
classes: the ubiquitin-processing protease (UBP) family and the
ubiquitin carboxyl-terminal hydrolase (UCH) family (25-27). Both UBPs
and UCHs are cysteine proteases containing in their active site
cysteine, aspartate, and histidine residues. While UCH amino acid
sequences are well conserved, UBPs share very limited homology (28).
Recently, a distinct family of cysteine proteases acting on
SUMO-1-conjugated substrates has been identified (28). The Apg4 family
may be considered as a novel subgroup of these cysteine proteases that
specifically cleaves the glycine-X bond at the COOH termini of
different GATE-16-like family members. Little is known about the
precise cellular function of UBPs and UCHs and the cellular mechanism
that regulates their activity. Here we showed that the cleavage
activity of recombinant HsApg4a is significantly lower than that found
in crude cytosol, suggesting that other cellular factors are involved
in the cleavage process. For example, HsApg4B may be required for
reconstitution of the full cleavage activity implying that the two
mammalian Apg4s act as a complex. Alternatively, the activity of Apg4A
may be regulated by other yet unidentified protein(s). Such regulation
of Apg4A activity may influence the function and possibly the
subcellular localization of GATE-16 and its other family members.
GATE-16 is the closest mammalian homologue of Aut7. We have
previously shown that Aut7p, similarly to GATE-16, interacts
genetically and physically with several v-SNAREs, which are involved in
ER to Golgi trafficking, and in vacuolar inheritance (29). GATE-16 has
been found to act at late stages of membrane fusion, coincidentally with the Golgi SNARE GOS-28 (7, 8). The precise mechanism by which
GATE-16 promotes fusion is yet unclear. It has been shown that GATE-16
binds preferentially to unpaired GOS-28 and inhibits GOS-28 binding to
its cognate t-SNARE syntaxin 5 (8). This activity of GATE-16 is similar
to the inhibition of syntaxin/VAMP binding by Munc18 (30) and the
Sed5p/Bet1p binding by Sly1p (31). In the case of GATE-16, it may act
as a SNARE protector that needs to be removed from GOS-28 prior to
fusion (2). However, GATE-16 may actively promote fusion. Accordingly,
the interaction with GOS-28 specifically localizes GATE-16 in a close
proximity to the sites of fusion. Hence, the potential of GATE-16 to be transiently lipidated, similarly to its yeast homologue Aut7p (20), may
serve to stimulate the formation a fusion pore during SNAREpin
formation and bilayer mixing. The activity of HsApg4a described in the
present work would be essential for such a process to occur.