From the Department of Medical Microbiology and
Immunology, University of Wisconsin-Madison Medical School, Madison,
Wisconsin 53706 and the ¶ Laboratory of Molecular Parasitology,
Rockefeller University, New York, New York 10021
Received for publication, January 10, 2001, and in revised form, March 9, 2001
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ABSTRACT |
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TbVCP is a member of the AAA
(ATPases Associated with a variety of cellular
Activities) family of proteins containing two ATPase
domains. Southern analysis indicates TbVCP to have a
single-locus, two-copy, genomic organization. One copy, but not both,
can be disrupted by targeted gene replacement, suggesting that
TbVCP is essential for trypanosome viability. Site-directed
mutagenesis of the ATP hydrolysis motifs indicates that the second
conserved ATPase domain is essential for TbVCP activity.
Constitutive overexpression of TbVCP with a single mutation
in the second hydrolysis motif or with mutations in both hydrolysis
motifs was not possible. Regulated overexpression of these mutants
resulted in cell death as a dominant negative phenotype. In each case
cell growth arrested at 24-h post-induction and at all stages of the
cell cycle as judged by replication of nuclear and kinetoplast genomes.
Onset of growth arrest coincided with the development of severe and characteristic morphological alterations for each mutant. Neither constitutive nor regulated overexpression of wild type
TbVCP or the single first hydrolysis domain mutant had any
overt effect on cell viability or morphology. However, the distinct
phenotype of the double mutant indicates that the first hydrolysis
domain, although not essential, does modulate overall TbVCP
function. Finally, yeast complementation studies demonstrated that
TbVCP can functionally replace the yeast homologue Cdc48p,
indicating that protein·protein interactions essential to
function have been maintained over great phylogenetic distances.
TbVCP is the first member of the
AAA1 family to be
characterized in African trypanosomes (1), a phylogenetically ancient lineage of protozoan parasites (2). Homologues of TbVCP are highly conserved over evolution and have previously been identified in
a wide range of organisms, including mammals (VCP, (3)), Saccharomyces cerevisiae (Cdc48p, (4)), Xenopus
laevis (p97, (5)), Arabidopsis thaliana (AtCdc48p,
(6)), as well as in Archaebacteria (7). These proteins have been
implicated in many cellular activities, including cell cycle regulation
(4), homotypic membrane fusion events (8-10), secretion (11),
endocytosis (12), cell division (6), and proteasome-mediated protein degradation (13, 14). TbVCP possesses many of the same
biochemical characteristics as the other homologues (1). It is a
homo-hexamer, each subunit having two copies of a conserved
~200-amino acid ATPase domain containing Walker A (ATP binding) and B
(ATP hydrolysis) motifs, and it has NEM-sensitive ATPase activity.
Other AAA family members having two conserved ATPase domains include
Pex1p and mammalian NSF (Sec18 in yeast) (15). Pex1p is essential for
peroxisomal biogenesis in S. cerevisiae (16). NSF plays an
essential role in the fusion cycle of transport vesicles with target
membranes in many steps of eukaryotic secretory pathways, including
endoplasmic reticulum to Golgi transport, intra-Golgi transport, and
synaptic fusion (17-19). During the process of vesicle and target
membrane fusion, NSF forms a 20 S complex with both SNAPs and
membrane-bound SNAREs derived from both the vesicle and target
membranes (20). Subsequent ATP hydrolysis by NSF is believed to mediate
conformational changes leading to disassembly of SNARE complexes in
post-fusion membranes (19).
The precise functions of VCP are less certain, but given its
sequence and structural relationship to NSF, it is believed to play an
analogous role in organellar biogenesis via homotypic membrane fusion
(8-10). This view is supported by evidence for interactions between
VCP homologues and resident SNAREs of the endoplasmic reticulum (21,
22) and Golgi (23). In mammalian cells these interactions are mediated
by a soluble adapter protein called p47, which functions analogously to
Evidence for VCP function in cell cycle control stems from work with
S. cerevisiae cdc48-1 mutants that arrest as large budded cells with an undivided nucleus in the bud junction (4). Because yeast
undergoes closed mitosis, Cdc48p must enter the nucleus to affect its
cell cycle-related function(s) and consequently contains a nuclear
localization signal (27). Interestingly then, AtCdc48p but not porcine
VCP can complement S. cerevisiae cdc48 mutants despite the
fact that both plants and animals have open mitosis (6, 27). However,
VCP failed to complement even when deliberately targeted to the yeast
nucleus (27) suggesting that VCP is unable to interact appropriately
with yeast Cdc48p accessory proteins across the fungi:vertebrate
phylogenetic divide.
The role of ATP hydrolysis in AAA protein function has been addressed
by site-specific mutagenesis of individual ATPase domains. Mutations of
the Walker A and B motifs in NSF indicate that the first ATPase domain
(D1) is responsible for the bulk of the NEM-sensitive ATPase activity
and that it is essential for NSF activity in in vitro fusion
assays (28, 29). Inactivation of the second domain (D2) had a modest
effect on the total ATPase and fusion activities. In contrast, when
this same strategy was employed with Pex1p, which functions in
peroxisome biogenesis, it was found that the second ATPase domain, but
not the first, was essential for activity in vivo (30). To
date no such analysis has been performed with any VCP homologue.
African trypanosomes are the causative agents of sleeping sickness in
humans and nagana in cattle. Our laboratory studies the cell biology of
these parasites focusing on secretory protein trafficking. In this
work, we further our previous studies (1) by performing an in
vivo functional analysis of TbVCP. Using a targeted
gene disruption strategy we demonstrate that TbVCP is an
essential gene for trypanosome viability. The nucleotide hydrolysis motifs of the two ATPase domains are inactivated by site-directed mutagenesis, and regulated expression of these mutants in transgenic trypanosomes is then used to assess the relative contribution of each
domain to in vivo TbVCP activity. Our results
indicate that the D2 domain is essential for TbVCP function
and that the D1 domain, although not itself essential, must augment the
D2 domain. Finally, yeast complementation experiments are performed to
establish that TbVCP can functionally replace S. cerevisiae Cdc48p. These studies provide novel structure/function
information on TbVCP as well as insights into its in
vivo functions.
Trypanosome Growth, Nucleic Acid Preparation, and Immunological
Procedures--
Trypanosoma brucei strain 427 procyclic
trypanosomes were utilized in targeted gene disruption experiments and
for constitutive expression of TbVCP ATP hydrolysis mutants
using the pKO vector series or the pXS2neo vector,
respectively (see below). Strain 29-13 procyclic trypanosomes (a strain
427 derivative) was used for regulated expression of TbVCP
mutants. Strain 29-13 (genotype: TETR, HYG,
T7RNAP, NEO) stably expresses the Tet repressor
under hygromycin selection and the T7 RNA polymerase under neomycin
selection. Transfection of this strain with reporter proteins in the
pLew100 vector (see below) allows tetracycline-inducible expression
from a procyclin promoter containing the Tet operator sequence (31).
Strain 427 procyclic trypanosomes were maintained in TMP medium at
27 °C as previously reported (32). pXS2 and pKO transformants of
this strain were selected with 50 µg/ml G418 and/or 25 µg/ml
hygromycin. Strain 29-13 was cultured at 27 °C in Cunningham's
medium (33) with 15% inactivated fetal bovine serum,
penicillin/streptomycin, 15 µg/ml G418, and 50 µg/ml
hygromycin. pLew100 transformants of strain 29-13 were selected with
2.5 µg/ml phleomycin. Trypanosomal DNA was prepared according to the
method of Medina-Acosta et al. (34). As needed, clonal cell
lines were established by limiting dilution in conditioned media (1:1
fresh and spent culture media supplemented to 20% fetal calf serum).
Metabolic radiolabeling, immunoprecipitation, and electrophoresis were
as previously described (32).
Synthetic Deoxyoligonucleotides--
The following synthetic
deoxyoligonucleotides were used for PCR. All sequences are from 5' to
3'. Sequences complementary to the target template are in
uppercase, added sequences are in lowercase,
restriction sites are underlined, and the altered nucleotide in mutagenic primers is in boldface and italic.
Primers for amplification of the 3'-flanking TbVCP genomic
targeting sequence were: JB165, aaaaaatctagataaCGCGGCGCTCGTCGCTC, 5'-primer (codons
727-732) with XbaI site; and JB166,
aaaaaagagctcCCCCCTCTCACTTTCC, 3'-primer (3'-UTR) with
SacI site. Primers for construction of DExx box (DB) mutants
were: JB149, GGTAGAGGTGGACCC, 5'-flanking primer (codons 155-160) for
DB1 1° and 2° amplification; JB155, GCCGAAGTTTTGGATTCC, 5'-flanking
primer (codons 428-433) for DB2 1° and 2° amplification; JB167,
ATTTTTATTGACCAAATAGACTC, 5'-internal primer (codons 290-297) for DB1 1° amplification; JB168,
GAGTCTATTTGGTCAATAAAAAT, 3'-internal primer (codons
290-297) for DB1 1° amplification; JB169,
CTTCTTCGATCAACTGGATTCCG, 5'-internal primer (codons 563-571) for DB2 1° amplification; JB170,
CGGAATCCAGTTGATCGAAGAAG, 3'-internal primer (codons
563-571) for DB2 1° amplification; JB171, CCCAGTCCACAACGGCC,
3'-flanking primer (codons 416-422) for DB1 1° and 2°
amplification; JB172, GACACCGTTCGCTCTTGC, 3'-flanking primer (codons
699-704) for DB2 1° and 2° amplification.
pKO Vector Construction and Targeted TbVCP Gene
Disruption--
A 2410-bp EcoRI/XbaI fragment
from pXS2 (35) containing 5'-3': 1) the procyclin
EP1-EP2 intergenic region, 2) the neomycin phosphotransferase gene (NEO), and 3) the tubulin
Wild type strain 427 procyclic trypanosomes
(TbVCP/TbVCP) were electroporated with either
HindIII/SacI-digested pKOneo Southern Analysis--
~3 µg of genomic DNA was separated
electrophoretically on 0.8% TAE-agarose gels and then
capillary-transferred to nitrocellulose membranes (Nitropure, Micron
Separations, Inc., Westboro, MA). Radiolabeled probe was generated by
random priming (High Prime DNA labeling kit, Roche Molecular
Biochemicals, Indianapolis, IN) utilizing [ DExx Box Mutant Construction--
The
HincII-KpnI region of the pBSIISK-polylinker was
first deleted by digestion and blunt-end religation. A 2459-bp
MluI/HincII fragment containing the entire
TbVCP gene (83 bp of 5'-UTR, 2343 bp of open reading frame,
33 bp of 3'-UTR) was then blunt-end-cloned from the genomic clone
p
Primary and secondary PCR reactions for the generation of DExx box (DB)
ATPase hydrolysis mutants were performed according to a previous study
(36) utilizing pfu DNA polymerase (Stratagene). Briefly,
overlapping primary PCR products were amplified from p
For the DB1 mutant, primary PCR reactions were performed utilizing
primer combinations JB149/168 and JB167/171; JB167 and JB168 encode the
mutated nucleotide. Secondary reactions were performed with JB149 and
JB171 as flanking primers. The secondary PCR product was digested with
AgeI and XhoI, and the 348-bp internal fragment
was inserted into AgeI/XhoI-digested pTbVCP to
generate pTbVCP(E294Q).
For the DB2 mutant, primary PCR reactions were performed utilizing
primer combinations JB155/170 and JB169/172; JB169 and JB170 encode the
mutated nucleotide. Secondary reactions were performed with JB155 and
JB172 as flanking primers. The secondary PCR product was digested with
StyI and NheI, and the 410-bp internal fragment
was inserted into NheI/StyI-digested pTbVCPXK.
The XhoI/KpnI fragment of pTbVCPXK was then
cloned into XhoI/KpnI-digested pTbVCP to generate
pTbVCP (E567Q). The same fragment was also cloned into
XhoI/KpnI-digested pTbVCP (E294Q) to generate
pTbVCP (E294Q/E567Q), i.e. the DB1/2 double mutant.
Constitutive and Inducible Expression of TbVCP ATP Hydrolysis
Mutants--
The insertion of wild-type TbVCP into the
constitutive expression vector pXS2neo has been described
previously (1). The entire DB1, DB2, and DB1/2 mutant TbVCP
coding regions were excised from their respective vectors with
HindIII/EcoRI and directionally cloned into the
corresponding sites of pXS2neo. Likewise, the wild type and
mutant coding regions were cloned into the
BamHI/HindIII sites of the inducible expression
vector pLew100 (31). The resulting plasmids series are designated
pXS2:WT, pXS2:DB1, etc., and pLew100:WT, pLew100:DB1, etc.
For constitutive expression, both wild-type
(TbVCP/TbVCP) and clonal single null
(TbVCP/
For inducible expression, strain 29-13 (NEO, HYG)
trypanosomes were electroporated with 10 µg of
NotI-linearized pLew100:WT, pLew100:DB1, pLew100:DB2, or
pLew100:DB1/2 by a modification of the standard protocol. After
electroporation the cells were transferred to 10 ml of conditioned TMP
media (1:1 fresh TMP and spent TMP supplemented to 20% fetal calf
serum) containing G418 (15 µg/ml) and hygromycin (50 µg/ml).
Transformants were selected on day 1 with phleomycin (2.5 µg/ml).
Viable transformants grew out in 2 weeks and were maintained initially
in conditioned media, and then in Cunningham's medium. In this system
homologous integration is targeted into the nontranscribed ribosomal
RNA spacer, a transcriptionally silent region of the trypanosome genome
(37). For induction of mutant TbVCP expression, tetracycline
was added to log phase cultures at a concentration of 0.5 µg/ml.
Microscopy--
Wild type and mutant TbVCP cell
cultures were seeded at 106/ml and induced with
tetracycline. Cells were harvested at 36-40 h post-induction, washed,
fixed, permeabilized, and stained with DAPI (1 µg/ml) as described
previously (1). Cells were viewed on a Zeiss Axioplan IIi at 100×
magnification. Differential interference contrast, and fluorescence
images were collected digitally and merged using OpenLabs 2.2 software
(Improvision Inc., Lexington, MA).
Yeast Complementation--
The yeast expression vector pFL61
(2µ plasmid) (38) containing the AtCDCc48 cDNA in the
sense (pAtCdc48s) and antisense orientations (pAtCdc48 TbVCP Is an Essential Gene--
We wished to determine if
TbVCP is essential for trypanosome viability by a targeted
gene disruption strategy. As trypanosomes are obligate diploids in
which genes are often found in tandem chromosomal arrays (41, 42), it
was first necessary to define TbVCP genomic organization.
The TbVCP genomic locus was mapped by restriction analysis
(Fig. 1A), and representative
single digests with endonucleases that cut once in and around the
TbVCP coding region are presented in Fig. 1B.
Each enzyme produces a single hybridizing band of unique size (except
ClaI and KpnI), suggesting that the
TbVCP gene is not tandemly reiterated and most likely has
two copies on homologous chromosomes. Detailed restriction analysis
indicates that there are no gross polymorphisms in the two
copies.2 This interpretation
of TbVCP genomic organization is supported by the results of
gene knockout experiments (see below).
Knockout vectors (pKOneo
Wild type and clonal cell lines transformed with either knockout
construct were analyzed by genomic southern hybridization (Fig.
1C). Probing ClaI- and
EcoRI-restricted wild type TbVCP/TbVCP DNA (TbVCP+/+) with a TbVCP 5'-UTR
probe revealed expected single fragments of ~5.7 and ~10 kb,
respectively (+/+, lanes 1 and 2). This probe hybridized to two distinct fragments in restricted DNA from
the TbVCP/
We attempted to generate null TbVCP double knockouts by
transformation of clonal TbVCP+/hyg trypanosomes
with pKOneo Constitutive Expression of TbVCP ATP Hydrolysis Mutants--
Like
other VCP homologues, TbVCP is an NEM-sensitive ATPase that
has two active domains containing Walker A and B ATP binding and
hydrolysis motifs (1). To assess the relative contribution of the two
ATPase domains to total TbVCP activity, as well as the
overall functional role of TbVCP in vivo, we
generated specific ATP hydrolysis mutants by a strategy that has been
previously used with mammalian NSF (28). Essential glutamic acid
residues in each Walker B hydrolysis motif (also called the DExx Box)
were mutated to glutamine creating the single ATPase mutants E294Q (DB1) and E567Q (DB2), and the double mutant E294Q/E567Q (DB1/2). Using
the constitutive expression vector pXS2neo (35), the wild
type and mutant TbVCP genes were electroporated into both
TbVCP+/+ and TbVCP+/hyg
procyclic cell lines, and transformants were selected under drug pressure.
Viable transformants were consistently generated in both wild type
TbVCP+/+ and single null
TbVCP+/hyg-recipient trypanosomes electoporated
with the wild type gene (Table II).
Viable transformants were also consistently generated with the DB1
mutant in both recipient strains. These transformants grew out within
the same period of drug selection (~1 week) as those transformed with
the wild type gene. However, viable transformants were rarely generated
when wild type trypanosomes were electroporated with the DB2 or DB1/2
TbVCP mutant genes and were never generated in the single
knockout recipients. The few wild type recipients (one for each
construct) that did survive drug selection took 3-4 times longer to
grow out, but then grew similarly to the wild type and DB1
transformants. A likely explanation for these neomycin-resistant cell
lines is that some genetic rearrangement occurred that eliminated or
inactivated the mutant gene without loss of the
NEO-selectable marker. Collectively, these results suggest
that overexpression of TbVCP with a mutated D2 ATPase domain
exerts a trans-dominant negative phenotype on endogenous
wild type TbVCP.
Inducible Expression of TbVCP Hydrolysis Mutants--
To examine
the phenotypic effect of these mutations we exploited a system for
regulated expression of toxic gene products in trypanosomes (31). The
strain 29-13 procyclic host cell line for this system is engineered to
constitutively express both T7 RNA polymerase and the Tet repressor.
The polymerase is expressed under neomycin selection by endogenous
read-through transcription in the tubulin locus. Tet repressor
expression is linked to hygromycin resistance and is driven by an
attenuated T7 promoter (from within its integration target site in the
RNP 1 locus). The inducible expression vector, pLew100,
contains both a wild type T7 promoter that drives expression of a
phleomycin resistance gene and a tetracycline-responsive procyclin
promoter for regulated expression of toxic gene products. The wild type
and mutant TbVCP genes were inserted into pLew100 and linear
plasmids were transfected into 29-13 host cells.
Clonal triple-resistant (neo, hyg, phleo) cell lines were selected and
analyzed for growth kinetics and TbVCP synthesis under tetracycline induction (Fig. 2). All
uninduced cell lines grew with normal kinetics, as did the wild type
and DB1 clones in the presence of tetracycline (Fig. 2A).
The DB2 and DB1/2 cell lines, however, ceased growth within 24 h
of induction concomitant with characteristic alterations in cellular
morphology by 36-40 h (see below). Over the ensuing 2-3 days, these
mutant cells became spherical with only the tip of the flagellum
protruding2 and by days 5 and 6 post-induction the cells
died. All cell lines overexpressed TbVCP polypeptides with
similar kinetics, and to the same extent, under tetracycline induction
(Fig. 2B). Elevated expression was not seen in uninduced
cells and quantitative enzyme-linked immunosorbent assays indicate that
maximum induction (at 24 h) typically reaches 3- to 5-fold over
levels of endogenous TbVCP.2 These results are
consistent with the failure to constitutively overexpress either the
DB2 or DB1/2 mutant and support the conclusion that when expressed they
exert a negative trans-dominant phenotype over endogenous
TbVCP. To further investigate this issue we measured the
stability of endogenous TbVCP in untransformed cells by
pulse-chase radiolabeling. Essentially no turnover was observed over a
48-h period (Fig. 2C), well beyond the onset of growth
arrest in the induced DB2 and DB1/2 cell lines. This confirms that the
phenotypes are trans-dominant in that they are manifested in
the continued presence of endogenous wild type protein.
Phenotypic Analysis of the TbVCP ATPase Mutants--
We first
assessed the effect of the DB1 and DB2 mutations on the ability of
TbVCP to oligomerize. VCP homologues from other systems are
known to be homo-hexamers, and our previous studies are consistent with
a similar oligomeric structure for TbVCP (1). Overexpression
of wild type and mutant proteins was induced in the 29-13 cell lines,
and the oligomeric state of TbVCP in cytosolic extracts was
determined by velocity sedimentation. Immunoblotting of gradient
fractions (Fig. 3) indicates that most of
the TbVCP in the wild type and mutant cell lines was
assembled into oligomers of the correct size (fractions 9-12). In each
case a lesser amount of TbVCP polypeptide sedimented at a
position intermediate to that expected for monomer and dimer. In no
case, however, did the amount of non-hexameric TbVCP in
mutant extracts differ significantly from that in the wild type
extract, or were significant amounts of TbVCP polypeptide
detected at the bottom of the gradients. These results suggest that
there are no gross defects in the ability of the ATP hydrolysis mutants
to properly oligomerize in vivo.
Trypanosomes have a characteristic morphology in which the flagellum
emerges via the posterior flagellar pocket, adheres along the length of
the cell body, and extends forward at the anterior end. The nucleus is
centrally located and the kinetoplast (the genome of the single
mitochondrion) is positioned near the base of the flagellum. Genome
replication and cell division occurs in a clear morphologically defined
order (43). Most cells in an asynchronously growing population are in
interphase, which is characterized by a single flagellum, kinetoplast,
and nucleus (1k/1n, ~85%). During cell division flagellar
duplication initiates first, followed closely by kinetoplast
replication generating intermediates with two kinetoplasts and a single
nucleus (2k/1n, <10%). Next nuclear division and flagellar
repositioning occurs to produce cells with two complete flagella,
kinetoplasts and nuclei (2k/2n, <10%). Ensuing longitudinal
cytokinesis produces two interphase progeny. Thus, the approximate
position within the cell cycle can be determined for any individual
cell by assessing organellar genome copy number.
Using these criteria we have evaluated the effect of overexpression of
TbVCP proteins on trypanosomal morphology and cell cycle
progression at an early period (36-40 h) when wild type cells were in
log phase growth but the mutants had ceased cell division. Induction of
wild type TbVCP altered neither the normal proportion (Fig.
4) nor the gross morphology of any stage
of the cell cycle (Fig. 5,
A-C). Likewise, the DB1 mutant remained normal in all
aspects following induction.2 Induction of the DB2 mutant,
however, led to growth-arrested forms with a characteristic
"spindle" morphology in which the cell body had apparently
retracted forward leaving a prominent posterior spine (Fig. 5,
D-I). In extreme cases the spine is completely devoid of
cytosol as judged by immunofluorescent imaging for HSP70.2
Arrested spindle cells were observed at all normal stages of the cell
cycle including 1k/1n, 2k/1n, and 2k/2n (Fig. 5, D-H), but
it was rare to find a cell arrested in the subsequent process of
cytokinesis. Interestingly, the percent of interphase cells was reduced
relative to wild type controls, and this was balanced by an increase in
products of aberrant cell division (Fig. 4, 1k/1n versus
other). The simplest of these were anucleate (1k/0n) cells (Fig.
5I) similar to the "zoids" generated by treatment with
anti-microtubule agents (44), but other multinucleate and/or multikinetoplastic forms were also seen (not shown). Such forms are
extremely rare in wild type cells.
The alteration of morphology with the double DB1/2 mutant was more
extreme than the DB2 phenotype (Fig. 6).
Again cells arrested in all normal stages of the cell cycle (Fig. 6,
A-C), but with a characteristic "tadpole" morphology in
which the cell body appeared retracted from the anterior end.
Consequently, tadpoles had rounded posterior cell bodies containing the
organellar genomes and narrow cytoplasm-containing anterior cell bodies
with adherent flagella. Like DB2, the proportion of cells arrested in
interphase was decreased relative to wild type, and the level of
aberrant division products was elevated (Figs. 4 and 6,
C-E). These included both products of uneven division,
anucleate zoids (1k/0n) and the matching 1k/2n cells (Fig.
6D), as well as other multigenomic (>2k/>2n) cells (Fig.
6E). But unlike DB2, many cells with fully replicated
organellar genomes also arrested in subsequent stages of cytokinesis
(Fig. 6, A and B). Most prominent in this
category were cells that arrested following complete longitudinal
fission. These cells presented as oppositely oriented daughters, each
with complete flagella and bodies containing segregated organellar
genomes but broadly fused across a transverse central plane (Fig.
6B).
Yeast Complementation--
In a final analysis we performed
complementation studies in S. cerevisiae to determine if
TbVCP could functionally replace the yeast homologue,
Cdc48p. The TbVCP genomic coding region was cloned into the
yeast expression vector pFL61 (38) in the sense and antisense
orientations. Controls for complementation included the pFL61 vector
alone and the pFL61 vector with the Arabidopsis homologue
AtCdc48 in both orientations. AtCdc48 has
previously been demonstrated to complement both cold- and
temperature-sensitive S. cerevisiae cdc48 conditional lethal
mutants (6). The S. cerevisiae cdc48-2 temperature-sensitive
strain MLY2006 was transformed with the pFL61 constructs, and
Ura+ transformants were selected at the permissive
temperature. Transformants were subsequently restreaked and challenged
at the non-permissive temperature (Fig.
7B).
Neither untransformed MLY2006 (Fig.7, sector a),
transformants carrying empty pFL61 vector (sector b), nor
transformants carrying either antisense gene (sectors c and
e) were able to grow at the non-permissive temperature. Both
sense TbVCP (sector f) and sense AtCdc48 (sector d) transformants were able to
grow at the non-permissive temperature, but the AtCdc48
colonies were smaller and took longer to develop than those carrying
TbVCP (longer incubation not shown). This finding is
consistent with the observation that AtCdc48 can complement
the cdc48-8 temperature-sensitive strain KFY189 at 36 °C
but not 37 °C (6). Two experiments were performed to confirm the
complementation analyses. First, the sense TbVCP plasmid was
re-isolated from two complementing transformants, verified by
restriction enzyme mapping, and then retransformed into MLY2006. The
transformants again grew at the non-permissive temperature (sector g) verifying that the TbVCP gene is
responsible for the complementation. Second, sense TbVCP
transformants that were grown in FOA, which causes the loss of
URA3-containing plasmids, lost the ability to grow at the
non-permissive temperature (sector h). These results
demonstrate that trypanosomal VCP is capable of functionally replacing
the yeast homologue.
Using African trypanosomes as a model system we have investigated
the in vivo functions of VCP/Cdc48p in eukaryotic cells. This protein is clearly essential for trypanosome viability as one, but
not both, copies of the endogenous gene can be knocked out by targeted
gene replacement. This is consistent with findings in S. cerevisiae where Cdc48p is essential for viability and growth of
haploid cells (4). Disruption of one endogenous copy of TbVCP had no effect on viability or morphology despite a
modest reduction of TbVCP protein as judged by immunoblot
analyses.2 Additional studies with single knockout
TbVCP+/hyg cells were performed to assess
whether reduction of TbVCP copy number had subtle effects on
secretory trafficking. Three reporter genes were introduced using the
stable constitutive expression vector pXS2neo (35). BiPN is
a soluble secreted reporter; BIPNMDDL is retained in the
endoplasmic reticulum; and 117wt is a
glycosylphosphatidylinositol-anchored cell surface reporter (35, 45).
Deletion of a single TbVCP copy had no effect on the
transport kinetics or localization of any of these reporters in the
TbVCP+/hyg genotype.2
Like many members of the AAA family, e.g. NSF and Pex1p,
TbVCP has two conserved ATPase domains. Specific mutagenesis
of the nucleotide binding and hydrolysis motifs in these domains
has been used to study the relative contribution of each to the overall function of both Pex1p (30) and NSF (28, 29). These studies indicate
that the Pex1p D2 domain and the NSF D1 domain are essential for
in vivo and in vitro function of these proteins.
Interestingly, sequence similarity between the essential domains of NSF
and Pex1p is greater than the similarity between the two domains within each individual protein (16). In contrast, both ATPase domains of
VCP/Cdc48p homologues are highly similar to each other, as well as with
the essential domains of NSF and Pex1p. We have used the same
mutagenesis approach to investigate the relative contribution of each
domain to TbVCP function in vivo. A single
critical glutamate residue within the DExx box sequence of each ATP
hydrolysis motif was changed to glutamine to create the DB1 (D1 domain)
and DB2 (D2 domain) single mutants, and the DB1/2 double mutant. The
analogous mutations in NSF reduced NEM-sensitive ATP hydrolysis by 69%
(D1), 28% (D2), and >90% (D1/D2) indicating that the activities of
the individual domains are additive and that the combined mutations can
completely ablate total ATPase activity (28). We have not directly
assayed ATP hydrolysis in our TbVCP mutants, but considering the effects of such mutations on NSF and the dramatic phenotypic alterations demonstrated herein, it is likely that these mutations significantly reduce the hydrolytic potential of each ATPase domain.
When the TbVCP mutants were independently transfected into
procyclic trypanosomes using a constitutive expression vector, no
transformants were obtained with either construct containing the DB2
mutation, whereas wild type and DB1 transformants were readily
generated. Given the low turnover rate of endogenous TbVCP (t1/2 > 48 h), this finding
suggests that the DB2 and DB1/2 mutants exert a
trans-dominant negative effect over endogenous wild type
TbVCP, thereby preventing outgrowth of transgenic cells. To
assess the phenotypes associated with the DB1 and DB1/2 mutants, we
resorted to expression in a regulated transcription system (31).
Induction of all constructs in clonal cell lines was rapid and
equivalent, leading to a 3- to 5-fold overexpression relative to
endogenous wild type TbVCP. As found with constitutive
overexpression, neither wild type nor DB1 TbVCP had any
effect on cell growth or morphology. Induction of the DB2 and DB1/2
mutants, however, resulted in complete cessation of cell growth within
24 h, much faster than the turnover of endogenous TbVCP
confirming that these mutants exert a trans-dominant
negative effect.
TbVCP is a homo-hexamer, and, if ATPase activity by one or
both domains is essential for assembly, it is possible that
overexpression of the mutants could inhibit oligomerization. This could
have several potential outcomes. Mutant TbVCPs may fail to
assemble leaving the endogenous wild type protein to form functional
homo-hexamers, a scenario that could account for the lack of phenotype
with the DB1 mutant. Alternatively, the mutant subunits may block
assembly of all TbVCP proteins, or may be included in mixed
hexamers with wild type subunits that are then dysfunctional in
downstream events mediated by TbVCP. Either of these
scenarios could account for the lethal phenotypes of the DB2 and DB1/2
mutants. However, when examined by velocity sedimentation no gross
defects in oligomerization were observed with any form of
TbVCP. Given the 3- to 5-fold overexpression of these
proteins, it is likely that all hexamers contain subunits of both
endogenous and induced TbVCP. Thus, although ATP hydrolysis by all subunits is clearly not required for oligomerization, hydrolysis by the 1-2 wild type subunits in each hexamer cannot be excluded as
essential for the assembly process. Nevertheless, our results indicate
that the trans-dominant effects of the mutants are likely exerted over some downstream function(s) of hexameric
TbVCP.
Following the arrest of cell growth (~24-h post-induction), both the
DB2 and DB1/2 mutants assumed characteristically altered cell
morphologies (DB2, spindles; DB1/2, tadpoles). Neither arrested in any
particular stage of the cell cycle (1k/1n, etc.), but in each case
there was a significant elevation in the number of cells with aberrant
mitochondrial and nuclear genomes. Given the number of cellular
processes in which other VCP homologues are known to participate, our
findings cannot illuminate the mechanism(s) by which the
TbVCP mutant phenotypes are affected in trypanosomes. However, several conclusions are possible. First, organellar genome profiles of arrested cells suggest that TbVCP is not a cell
cycle regulator per se. Were this the case we would expect
cells to arrest at a specific stage of organellar replication. The
yeast gene, CDC48, was originally identified as a cell
division cycle mutant (46), and this is one of the many functions
typically assigned to its homologues. However, the unique features of
trypanosome organellar replication allow a direct assessment of the
cell cycle, and our results argue against a direct function in this
process, at least in trypanosomes. Second, the elevated level of
aberrant genomes must result from either unequal segregation of
daughter organelles (1k/0n and 1k/2n progeny), from cytokinesis without organellar replication (1k/0n and 0k/1n progeny), or from ongoing organellar replication without cytokinesis (3k/3n, etc). Such aberrations are never observed in normal cells, suggesting that TbVCP may have a role, direct or indirect, in cytokinesis.
Finally, the fact that the DB1/2 mutant has a subtle but distinct
phenotype relative to DB2 alone suggests that there is a hierarchy of
TbVCP functions in which D1 hydrolytic activity may be
required. Such a conclusion would not be obvious from the apparent lack
of phenotype when the DB1 mutant is expressed in isolation. Thus,
although the D2 domain seems to be paramount in TbVCP
activity, the D1 domain must also contribute to overall function
(discussed below).
VCP/Cdc48p homologues have been implicated in many other cellular
functions, the best characterized of which are proteasome activity (13,
14, 25) and homotypic membrane events, such as endoplasmic reticulum
fusion (10, 22) and Golgi fusion (8, 9). In each case, the ATPase has
been shown to physically interact with either soluble (13, 14, 24, 25)
or membrane-bound (21, 22, 23) cofactors and substrates for
ATP-dependent folding/dissociation reactions. Molecular
chaperone activity may in fact be the common feature of all VCP/Cdc48p
functions, and there are at least two soluble accessory proteins that
bind to and regulate its activity in a mutually exclusive manner,
i.e. p47 in homotypic fusion and Ufd1/Npl4 in proteasomal
function (24, 25). It has been proposed that additional cofactors will be associated with other specific activities accounting for the ability
of a single ATPase to mediate multiple functions (25). Presumably,
these functions require precisely co-evolved protein·protein interactions among the ATPase and its various cofactors and substrates. It is surprising then that the TbVCP gene is able to
complement a conditionally lethal yeast cdc48 mutant.
Whether TbVCP can act as a global surrogate in yeast is not
clear; we can only be certain that we have complemented the essential
Cdc48p function(s) that are compromised in this particular mutant.
Nevertheless, trypanosomes and fungi are widely divergent (2) and,
despite 64% identity between the two homologues (1), it is remarkable
for complementation to occur when multiple protein·protein
interactions are required.
There is an emerging concept of how VCP homologues may function as
force-generating motors. This view is perhaps best illustrated by the
role of the mammalian homologue p97 and its cofactor p47 in homotypic
membrane fusion. Fusion is thought to be driven by the interaction of
SNARE proteins in opposing membranes after which p97·p47 complex
mediates the ATP-dependent disruption of paired SNAREs in
the post-fusion membrane. The complex would then dissociate leaving the
free SNAREs available for further rounds of fusion. Indeed, p97·p47
has been shown to interact with the t-SNARE syntaxin 5 in both
endoplasmic reticulum and Golgi membranes (22, 23). Furthermore,
binding is mediated by p47 (23) and after in vitro fusion,
p97·p47 is released from membranes (22). Recent crystallographic and
cryoelectronmicroscopy studies suggest a detailed structure for
the p97·p47 complex (47, 48). p97 monomers assemble as two stacked
concentric rings composed of hexamers of the D1 and D2 sub-domains with
six p47 subunits associated around the periphery. Binding of either ATP
or ADP results in a conformational change in the core p97 structure. In
the absence of nucleotide, or with bound ADP, p47 association is loose
and mobile. But, strikingly, in the presence of ATP the p47 monomers project radially in a constrained manner. Overall, these findings suggest a model in which ATP binding induces a strained conformation in
p97 that pushes p47 monomers outward from the central axis, which in
turn could generate the force required to disassemble a t-SNARE pair
attached to any two p47 monomers within the complex (48). Subsequent
ATP hydrolysis would restore the relaxed conformation followed by
release of p97·p47 from membranes.
Our findings with TbVCP mutants have significant
implications for this model. The D2 hydrolysis motif is absolutely
essential for in vivo TbVCP function and,
although not essential, the D1 hydrolysis motif apparently modulates
overall activity. Thus, ATP hydrolysis by the D2 domain is likely to be
central to the observed conformational changes in the p97·p47
complex. Zhang et al. (47) have speculated that ATP
hydrolysis in one domain promotes nucleotide exchange in the other, and
that subsequent hydrolysis by the second domain promotes nucleotide
exchange in the first, producing a ratchet-like conformational cycle
for force generation in the p97 core. Our results argue against an
essential role for ATP hydrolysis by the D1 domain in such a model, but a role for this domain in overall function cannot be discounted. Because it is bound ATP that induces the strained conformation in the
p97·p47 complex (48), reversible nucleotide binding in the D1 domain
may contribute to, or even be essential for, the strained-relaxed
oscillation. Perhaps the affinity of the D1 binding site is low enough
to allow reversible ATP binding without hydrolysis, or alternatively,
the full hydrolysis/exchange cycle in the neighboring D2 domain may
regulate ATP exchange in the D1 domain as suggested by Zhang et
al. (47). In either scenario, ATP hydrolysis in the D1 domain,
although not required, would enhance normal nucleotide exchange thereby
accounting for the observed phenotypic differences between the DB2 and
DB1/2 TbVCP mutants.
Obviously, proof and/or refinement of these models will require further
work. Important issues that remain are whether both ATP binding sites
must be occupied for conformational change, at what point nucleotide
exchange occurs, and whether bound nucleotide is required for
association of p97·p47 with SNARE complexes. Addressing these issues
will require more structural studies utilizing mutant proteins such as
we have generated. In addition, the establishment of
trans-dominant mutants in an inducible trypanosomal
expression system provides unique opportunities for further research
into TbVCP function. In particular, with the loss of ATPase
activity it may be possible to lock TbVCP in a bound
conformation leading to identification of other essential protein
cofactors and substrates in trypanosomes. Given the high sequence
conservation of AAA proteins and the ability to complement genetically
over wide phylogenetic distances, it is likely that any new findings
will extend to other VCP homologues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SNAP in the formation of NSF 20 S complexes (24). In addition, a
complex of cytosolic proteins involved in ubiquitin-mediated proteasome
function (Ufd1) and nuclear import (Npl4) have been shown to physically
interact with the VCP homologue p97 (25). The growing view is that VCP is a multifunctional protein that affects different activities via
specific adapter proteins (25). The commonality of these functions may
be the ATP-dependent protein folding/unfolding activity of
VCP as has been demonstrated with the archaeal homologue (26).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
intergenic region was cloned into the corresponding sites in
pBluescript IISK
(pBSIISK
, Stratagene, San Diego CA) to generate
pKOneo. Next the NEO cassette was replaced with
a hygromycin phosphotransferase gene cassette (HYG) using
flanking AscI/PacI sites (a generous gift of Dr.
Ian Manger, Stanford University) to create pKOhyg. Specific
TbVCP disruption constructs were assembled by first inserting a 349-bp HindIII/NheI 5'-flanking
TbVCP genomic fragment (nucleotides
353 to
10 relative
to nucleotide +1 of the TbVCP open reading frame) into
corresponding sites in the upstream polylinker portion of the pKO
vectors. A 455-bp fragment was then amplified (primers JB165/166) from
the 3'-flanking TbVCP genomic region and inserted into
XbaI/SacI sites in the downstream polylinker portion of the pKO vectors. Digestion of these constructs
(pKOneo
vcp and pKOhyg
vcp) with
HindIII/SacI releases linear fragments bearing
distinct drug selectable markers flanked by homology sequences for
complete targeted replacement of the genomic TbVCP alleles.
vcp (5 µg) or pKOhyg
vcp (50 µg) by standard protocol (35).
Transformants were cultured, and appropriate drug selection was applied
on day 2; stable transformants grew out within 2 weeks and heterozygous
TbVCP/
tbvcp lines were cloned by
limiting dilution. The double null disruption was attempted by
electoporation (n = 5) of a single null clone
(TbVCP/
tbvcp::hyg) with 5 µg of HindIII/SacI-digested
pKOneo
vcp. Wild type trypanosomes were simultaneously
electroporated (n = 3) as a positive control. At 2 days, wild-type recipient trypanosomes were selected with G418
(n = 3); and single null recipients were selected with
either G418 (n = 1) or G418 and hygromycin
(n = 4).
-32P]dCTP
(PerkinElmer Life Sciences, Boston, MA) according to the manufacturer's instructions. For determination of wild-type genomic organization, the 305-bp pVCP insert (1) was used as probe template;
and for verification of the TbVCP single null
disruptions, either the TbVCP 5'-UTR, or the
NEO or HYG coding regions were used as probe
template. Membranes were prehybridized (QuikHyb, Stratagene, San Diego,
CA) for 30
60 min at 68 °C followed by a 2-h hybridization
at 68 °C. High stringency final wash was 0.1× SSC, 0.1% SDS,
60 °C. Hybridization was visualized by autoradiography.
5VCP (1) into the EcoRV site of pBSIISK (
HincII-KpnI) to generate pTbVCP. The
XhoI-KpnI fragment of pTbVCP was next cloned into
XhoI/KpnI-digested pBSIISK
to generate pTbVCPXK.
5VCP in two
separate reactions. Each reaction utilized a unique flanking primer and
a complementary mutagenic primer to introduce a single altered codon (E
Q) in the overlapping ends of the paired primary products.
Secondary reactions utilized 10 ng each of the primary PCR products as
templates with the two unique flanking primers. In the secondary
reactions, denatured primary products re-anneal at the overlapping ends
that contain the mutated codon and are amplified as one contiguous
product. This process was performed independently for the two
TbVCP ATPase domains, and each final product was sequenced
though the region of PCR amplification to verify that only the desired
mutation was generated.
tbvcp::hyg)
strain 427 trypanosomes were electroporated with
BstXI-linearized pXS2:WT (n = 2), pXS2:DB1 (n = 3), pXS2:DB2 (n = 3), or
pXS2:DB1/2 (n = 3) by standard protocols (35). At 2 days, wild-type recipient trypanosomes were selected with G418, and
single null recipients were selected with G418 and hygromycin. The
entire experiment was performed twice for the wild-type recipients and
three times for the single null recipient. In all cases homologous
integration was targeted to the tubulin locus.
s) (6) was
kindly provided by Dr. Sebastian Bednarek (University of
Wisconsin-Madison), as was the haploid S. cerevisiae strain
MLY2006 (ts cdc48-2 ura3-52) (21). To generate sense
(pTbVCPs) and antisense (pTbVCP
s) constructs for expression in yeast
the TbVCP-containing MluI/HincII
genomic fragment was blunt-end-ligated into pFL61, and clones in both
orientations were selected. Strains MLY2006 was transformed with each
construct by the LiAc, polyethylene glycol transformation method (39). Ura+ transformants were selected on synthetic minimal media
minus uracil at the permissive temperature (24 °C). The
Ura+ transformants were restreaked onto separate YPD plates
and incubated at either the permissive or the non-permissive
temperatures (37 °C). To confirm the identity of the plasmids that
complemented the conditional growth defects of MLY2006, the pTbVCPs
construct was re-isolated according to a previous method (40). The
isolated plasmid was verified by restriction enzyme mapping and
subsequently retransformed into MLY2006 and challenged at the
non-permissive temperature. In addition, positive transformants were
restreaked onto synthetic minimal media containing 0.005% uracil
(Sigma), 0.1% 5-fluoro-orotic acid (Sigma) to cause selective loss of
the URA3-containing construct. Isolated colonies that grew
on FOA were then restreaked onto YPD media and incubated at the
non-permissive temperature.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Targeted gene disruption of TbVCP.
A, restriction map of the TbVCP genomic locus
(top) and the HindIII/SacI-linearized
knockout constructs (bottom). The positions of the
TbVCP gene (black box), the NEO and
HYG genes (gray box), the procyclin
(ProInt) and tubulin (TubInt) intergenic regions
(white boxes), and probes used in Southern hybridization
(hatched boxes) are denoted. Regions of targeted integration
are indicated. Restriction endonuclease sites are as follows:
A, AscI; B, BstEII;
C, ClaI; E, EcoRI;
H, HindIII; K, KpnI;
N, NheI; Ps, PstI;
S, SacI; SII, SacII;
P, PacI; Xb, XbaI;
X, XhoI. B, genomic Southern
hybridization of wild type strain 427 procyclic DNA using an internal
coding region PCR probe. The scale refers to size in kilobase pairs.
C, sequential Southern hybridization of ClaI- and
EcoRI-digested genomic DNA from wild type
TbVCP/TbVCP (+/+),
TbVCP/ tbvcp::neo
(+/neo), and
TbVCP/
tbvcp::hyg (+/hyg)
clones with specific 5'-UTR (lanes 1-6),
NEO (lanes 7-12), or HYG
(lanes 13-18) probes. A shorter exposure of lanes
1 and 2 is presented for clarity. Scales
refers to size in kilobase pairs.
vcp, pKOhyg
vcp)
were assembled containing NEO or HYG genes
flanked by native sequences for robust RNA processing (procyclin and
tubulin intergenic regions), which in turn were flanked by homologous
targeting sequences for TbVCP locus (Fig. 1A).
Electroporation of the excised HindIII/SacI
fragment leads to targeted gene replacement of an entire
TbVCP gene by homologous recombination. Expression of the
selectable marker is mediated by endogenous read-through transcription.
tbvcp::neo
(TbVCP+/neo, lanes 3 and
4) and
TbVCP/
tbvcp::hyg
(TbVCP+/hyg, lanes 5 and
6) clones. In each case, one fragment is the same size as
wild type, and the second is either larger (~6.0 or ~6.2 kb,
ClaI, lanes 3 and 5) or smaller
(~6.5 kb, EcoRI, lanes 4 and 6).
Each new fragment matches the predicted size for the desired homologous
integration event consistent with the loss of a single TbVCP
copy due to targeted gene replacement. Furthermore, the equal
signal intensity of the wild type and integrant fragments support the
presence of just two homologous copies of TbVCP in the wild
type genome. To verify that the new bands were generated by targeted
replacement of a wild type copy, the blot was sequentially stripped and
hybridized with NEO- and HYG-specific probes.
Again in each case, the probes hybridized with predicted fragments in the corresponding DNA only (TbVCP+/neo,
lanes 9 and 10;
TbVCP+/hyg, lanes 17 and
18).
vcp; wild type
TbVCP+/+ cells were used as a positive control
for transformation (Table I). All wild
type recipients were selected with neomycin, and the
TbVCP+/hyg recipients were selected with either
neomycin, or neomycin and hygromycin. As expected, viable
neomycin-resistant transformants grew out of all wild type recipients,
and Southern analysis confirmed that a single TbVCP copy was
replaced. Viable transformants also grew out following selection of
TbVCP+/hyg recipients with neomycin alone.
However, challenge of these transformants with hygromycin resulted in
cell death, and Southern analysis indicated that the
neomycin-selectable marker had replaced the hygromycin-selectable
marker.2 Simultaneous neomycin and hygromycin selection of
TbVCP+/hyg recipients resulted in no viable
transformants. Collectively, these results establish that there are two
homologous copies of TbVCP and that one copy, but not both,
can be disrupted without effecting cell viability.
Attempted double TbVCP gene disruption
Constitutive expression of TbVCP hydrolysis mutants
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Fig. 2.
Regulated expression of TbVCP ATP
hydrolysis mutants. Log phase cultures of clonal 29-13 procyclic
cell lines bearing wild type and mutant forms of TbVCP in
the pLew100 vector were subcultured at 2.5 × 105/ml
in the presence or absence of 500 ng/ml tetracycline. A,
growth kinetics of wild type (squares), DB1
(circles), DB2 (diamonds), and DB1/2
(triangles) clones were determined by counting motile cells
in a hemocytometer. Open symbols, control uninduced
cultures; filled symbols, tetracycline-induced cultures.
B, whole cell lysates from tetracycline-induced cultures
prepared at the indicated times post-induction were fractionated by
SDS-polyacrylamide gel electrophoresis (5 × 106 cell
equivalents per lane) and transferred to filters. Blots were probed
with anti-TbVCP antibody (upper panel) and then
reprobed with anti-BiP antibody (lower panel) as a loading
control. C, untransformed procyclic cells were
pulse-radiolabeled with [35S]methionine/cysteine for 15 min and then chased for 48 h. At the indicated times, cell
extracts were prepared from equivalent volumes of cell culture (0.5 ml); and total radiolabeled TbVCP polypeptides were
immunoprecipitated with specific antibody. A scan of a 5-day exposure
is presented.
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Fig. 3.
Oligomerization of TbVCP ATPase
mutants. At 24-h post-induction, cytosolic extracts of the clonal
strain 29-13 cell lines were prepared and fractionated by sucrose
gradient velocity sedimentation as described previously (1). Gradient
fractions were analyzed by SDS-polyacrylamide gel electrophoresis and
immunoblotting with anti-TbVCP antibody. Representative
gradients are presented for wild type, DB1, DB2, and DB1/2
TbVCP. Fraction numbers increase from top to
bottom of the gradient, and in all cases the
bottom-most fraction is presented. The sedimentation
positions of internal size markers are indicated for each gradient as
follows: b, bovine -globulin (170 kDa); c,
catalase (250 kDa); a, apoferritin (460 kDa), t,
thyroglobulin (660 kDa).
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Fig. 4.
Organellar genome content of inducible
TbVCP cell lines. At 36-40 h post-induction wild
type, DB2, and DB1/2 cell lines were fixed and stained with DAPI.
Individual cells were scored for number of kinetoplasts (k)
and nuclei (n) by fluorescence microscopy. Data are
presented as mean percent (± S.E.) of total cells counted (>200) for
three individual experiments. Cells with zoid phenotypes (1k/0n) are
also included in the "other" category. No wild type cells were
observed in the other and zoid categories.
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Fig. 5.
Morphology of wild type and DB2 mutant cell
lines. Log phase wild type (A-C) and DB2
(D-I) mutant inducible cell lines were fixed at 40-h
post-induction and stained for DNA with DAPI (blue).
Individual cells were examined by differential interference contrast
and fluorescence microscopy as described under "Experimental
Procedures"; digitally merged images are presented. The status of
organellar genome replication is indicated for each cell; and the
normal positions of kinetoplast (k) and nuclear
(n) genomes are indicated for each wild type cell. All cells
are presented with the anterior end oriented toward the
top.
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Fig. 6.
Morphology of the DB1/2 mutant cell
line. Log phase-inducible DB1/2 mutant cells were prepared for
microscopy as described in Fig. 5. The status of organellar genome
replication is indicated for individual cells.
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Fig. 7.
Complementation of a S. cerevisiae
cdc48 temperature-sensitive mutant. The following cell lines
were grown at the permissive (A, 24 °C) and
non-permissive (B, 37 °C) temperatures: untransformed
host strain MLY2006 (a); MLY2006 transformed with the pFL61
vector (b); or MLY2006 transformed with pFL61-containing
antisense AtCdc48 (c), sense AtCdc48
(d), antisense TbVCP (e), and sense
TbVCP (f) genes. As complementation controls,
plasmid was recovered from sense TbVCP transformants and
used to retransform MLY2006 cells (g), and a MLY2006 sense
TbVCP transformant was cured of its plasmid by negative
selection in FOA (h).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are indebted to Drs. Sebastian Bednarek, Anant Menon, and Martin Latterich for providing reagents and/or thoughtful discussion and comments. We are particularly grateful to Dr. Sebastian Bednarek for critical reading of the manuscript.
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FOOTNOTES |
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* This work supported in part by National Institutes of Health Grant AI35739 (to J. D. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Institutes of Health Cellular and Molecular Parasitology Training Grant AI07414. Current address: Dept. of Microbiology and Immunology University of Rochester, 601 Elmwood Ave., Box 672, Rochester, NY 14642.
A Burroughs Wellcome Fund New Investigator in Molecular
Parasitology. To whom correspondence should be addressed: Dept. of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-3110; Fax: 608-262-8418; E-mail:
jdbangs@facstaff.wisc.edu.
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M100235200
2 J. R. Lamb, V. Fu, E. Wirtz, and J. D. Bangs, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: AAA, ATPases Associated with a variety of cellular Activities; VCP, valosin-containing protein; NSF, NEM-sensitive protein; SNAP, soluble NSF accessory protein; SNARE, SNAP receptor; NEM, N-ethylmaleimide; FOA, 5-fluoro-orotic acid. PCR, polymerase chain reaction; UTR, untranslated region; DB, DExx box; bp, base pair(s); DAPI, 4',6-diamidino-2-phenyl-indole; kb, kilobase(s); FOA, 5-fluoro-orotic acid.
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