From the Division of Molecular Transport, Department of Internal
Medicine, and the Howard Hughes Medical Institute,
University of Texas Southwestern Medical Center,
Dallas, Texas 75235
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The vacuolar type proton-translocating ATPase of clathrin-coated vesicles is composed of two large domains: an extramembranous catalytic sector and a transmembranous proton channel. In addition, two polypeptides of 50 and 57 kDa have been found to co-purify with the pump. These proteins, termed SFD (sub-fifty-eight-kDa dimer) activate ATPase activity of the enzyme and couple ATPase activity to proton flow (Xie, X.-S., Crider, B.P., Ma, Y.-M., and Stone, D. K. (1994) J. Biol. Chem. 269, 28509-25815). It has also been reported that the clathrin-coated vesicle proton pump contains AP50, a 50-kDa component of the AP-2 complex responsible for the assembly of clathrin-coated pits, and that AP50 is essential for function of the proton pump (Liu, Q., Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 31592-31597). We demonstrate through the use of anti-AP50 antibody, identical to that of the latter study, that hydroxylapatite chromatography removes AP50 from impure proton pump preparations and that purified proton pump, devoid of AP50, is fully functional.
To determine the true molecular identity of SFD, both the 50- and
57-kDa polypeptides were directly sequenced. A polymerase chain
reaction-based strategy was used to screen a bovine brain cDNA
library, yielding independent full-length clones (SFD-4A and SFD-21);
these were identical in their open reading frames and encoded a protein
with a predicted mass of 54,187 Da. The SFD-21 clone was then used in a
reverse transcription-polymerase chain reaction-based strategy to
isolate a related, but distinct, transcript present in bovine brain
mRNA. The nucleotide and predicted amino acid sequences of this
isolate are identical to SFD-21 except that the isolate contains a
54-base pair insert in the open reading frame, resulting in a protein
with a predicted mass of 55,933 Da. Both clones had 16% identity to
VMA13 of Saccharomyces cerevisiae. No sequence
homology between the SFD clones and AP50 was detectable. Anti-peptide antibodies were generated against an epitope common to the
two proteins and to the unique 18-amino acid insert of the larger
protein. The former reacted with both components of native SFD, whereas
the latter reacted only with the 57-kDa component. We term the 57- and
50-kDa polypeptides SFD and SFD
, respectively.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Vacuolar, or V-type, proton translocating ATPases are found in diverse eukaryotic endomembrane compartments and plasma membranes of epithelia, macrophages, and specialized polarized cells. Their functional roles reflect this broad distribution and range from processing receptor-ligand complexes in endosomes to osteoclast-mediated bone dissolution (1-3).
V-type proton pumps are complex hetero-oligomers with peripheral and intramembranous domains. Preparations of the proton pump of clathrin-coated vesicles contain at least nine proteins of 116, 70, 58, 57, 50, 40, 39, 34, and 33 kDa, as well as four smaller polypeptides with apparent molecular masses of 10-17 kDa (4-7). This polypeptide composition is similar to that of most V-type pumps, reflecting intense phylogenetic conservation (8-12). Functional domains of the coated vesicle proton pump include VB,1 a proton channel that is probably composed of the 116-, 39-, and 17-kDa components (5), and VC, the catalytic sector of the complex consisting of a set of polypeptides of 70, 58, 40, 34, 33, 15, 14, and 10 kDa (6). Separation of VC from the holoenzyme results in marked changes in enzyme activity; although native enzyme hydrolyzes Mg-ATP at a rate 3-fold higher than Ca-ATP, isolated VC can hydrolyze ATP in the presence of Ca2+, but not Mg2+, and cannot support ATP-driven, vectorial proton movement (7). Recently, we reassembled purified VB and VC to yield a complex with Mg-ATPase and proton pumping activities similar to those of native enzyme. Essential to this reconstitution was a heretofore unrecognized 50-57-kDa polypeptide heterodimer, which we termed SFD, for sub-fifty-eight-kDa dimer (6).
Others recently reported that the 50-kDa subunit of the AP-2 clathrin assembly complex is present in their preparation of the clathrin-coated vesicle proton pump and that AP50 might function as a kinase, catalyzing autophosphorylation as well as phosphorylation of the 58-kDa subunit of the pump (13). Subsequently, it was reported that AP50 was essential for the function and assembly of the clathrin-coated vesicle proton pump (14). Although such an interaction could explain the basis whereby endomembrane acidification is regulated by an organellar specific mechanism, these reports are not easily reconciled with the prior demonstrations that AP-2 (and its component, AP50) is found only in plasma membrane-associated clathrin-coated pits and vesicles (15, 16) and that clathrin-coated vesicles associated with the plasma membrane cannot acidify their interiors (17, 18).2
To investigate this point, and to identify the 50- and 57-kDa
polypeptides of SFD and their role(s) in pump function, we studied the
possible relationship of the 50-kDa component of SFD to AP50. Recently,
we reported that phosphorylation of 50- and 58-kDa polypeptides was
observed only with impure clathrin-coated vesicle pump preparations and
that purified pump required SFD for function but lacked any kinase activity (6). (This observation, however, did not exclude the
possibility that the 50-kDa component of SFD is AP50, since the primary
structure of the latter (19) lacks any known nucleotide binding motif,
and its role as a kinase is conjectural.) We now demonstrate that AP50
itself is not a component of the purified clathrin-coated vesicle pump;
nor is it required for ATP-driven proton translocation. Through direct
protein sequencing and molecular cloning, we show that the 50- and
57-kDa components of SFD are highly related proteins that probably
arise through an alternative splicing mechanism. These subunits are
termed SFD (57 kDa) and SFD
(50 kDa). They share 16% identity to
a 54-kDa polypeptide of the yeast vacuolar proton pump encoded by the
VMA13 gene (20). No homology, however, exists between these
proteins and AP50 or any AP isoforms. These studies demonstrate that
SFD, and not AP50, is required for function of the vacuolar-type proton
pump of clathrin-coated vesicles.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparations and Materials--
Isolation of clathrin-coated
vesicles from bovine brains (21) and preparations of liposomes from
purified lipids (22) were performed as reported. Purification of the
proton-translocating ATPase of clathrin-coated vesicles was performed
by sequential solubilization with C12E9,
hydroxylapatite (HTP) chromatography, (NH4)2SO4 fractionation, and
glycerol gradient centrifugations (4); purified H+-ATPase
had a specific activity of 14-16 µmol Pi·mg
protein1·min
1. Purified proton pump was
depleted of SFD by treatment with Zwittergent 3-16, followed by
glycerol gradient centrifugation, as reported (6). Purified AP-2 (µ2)
complex from rat brain and chicken anti-AP50 (µ2) IgY were the
generous gifts of Dr. Thomas Kirchhausen (Harvard University). Goat
anti-chicken IgY antibody was obtained from Pierce, HTP (Bio-GEL HTP)
was from Bio-Rad; restriction enzymes, T4 DNA sequencing
ligase, and a nick translation kit were from Boehringer Mannheim; DNA
sequencing reagents and enzymes and the GeneAmp PCR reagent kit were
from Perkin-Elmer; Escherichia coli strains XL1-Blue and BB4
and helper phages R408 were from Stratagene; radioactive reagents were
from Amersham Corp.; nitrocellulose membranes for plaque lift were from
Millipore Corp.; and chemicals for SDS-PAGE were from Bio-Rad. A bovine
brain cDNA library was the generous gift of Dr. Richard Dixon,
University of Texas Health Science Center at Houston. The sources of
other materials used in this study have been identified previously
(3-7).
Protein Sequencing-- About 200 pmol of each of the 50- and 57-kDa components of SFD were separated by SDS-PAGE (12% acrylamide), and proteins were electrophoretically transferred to Immobilon polyvinylidene difluoride filters, from which the 50- and 57-kDa polypeptide bands were excised, and digested with trypsin or LysC in situ (23). Released peptides were separated by reverse phase high performance liquid chromatography using a 2.1 × 150-mm RP300 column from Perkin-Elmer and were subjected to automated Edman degradation using a model 477A amino acid sequencer from Applied Biosystems with the manufacturer's standard program and chemicals.
Synthesis of a DNA Probe by PCR-- To obtain a DNA probe for screening a bovine cDNA library, four rounds of PCR were performed. The first PCR was carried out with a bovine cDNA library as template, using two primers (I, 5'-GCIGCIGCIGT(T/C)TGIGG(T/C)TG(T/C)TC-3'; II, 5'-GTIATIGAICA(G/A)(C/T)TIGGIGGIAA-3') based upon primary peptide sequence. After 30 cycles of PCR, this reaction generated one dominant band of ~500 bp, but sequencing revealed that this arose from primer I annealing to two separate sites in a transcript that did not encode amino acids flanking those used to design primer I. Under the premise that a genuine, relevant PCR product had also been produced (at a much lower copy number than the ~500-bp product), a second PCR reaction was performed. The reaction mix was diluted 100-fold, and 1 µl of the diluted reaction mixture was used as template in this second PCR reaction using primer II and primer III (III, 5'-TATTCCCA(A/G)TT(A/G)TGIACCATIA(A/G)(T/C)TT-3') that was designed according to an expressed sequence tag clone (TO6491) identified by screening expressed sequence tag data bases with peptide sequences obtained from the direct sequencing of the 50-kDa component of SFD. This second round of PCR amplified a 120-bp fragment. To generate a larger DNA fragment suitable for cDNA library screening, the sequence of the 120-bp fragment was used to design two additional primers (IV, 5'-AGACTGGAGTTGTTTACCAAGATA-3'; V, 5'-CTTCTGTACAGCCAGGAGGGCGTT-3'). Primer IV and T3 promotor primer were used in the third round of PCR, using bovine brain cDNA library as template. The reaction mix was diluted 100-fold, and 1 µl of the diluted reaction mixture was used as template in the final round of PCR using primer V and T3 promotor primer. This round of PCR generated an 856-bp fragment that was used to screen the library.
Cloning of cDNAs Encoding SFD of Clathrin-coated Vesicle
ATPase--
The 856-bp PCR fragment was labeled with
[-32P]dCTP by nick translation and used to screen a
bovine brain cDNA library in
ZAP, which had been transfected
into E. coli strain BB4. Plaques were transferred to
nitrocellulose membranes by a double-lift procedure (26). The membranes
were then prehybridized for at least 4 h at 60 °C in a solution
containing 5 × SSC, 5 × Denhardt's solution, 0.1 mg/ml
sheared salmon sperm DNA, and 0.1% SDS. Hybridization was performed at
60 °C overnight with the same solution plus labeled probe, which was
added at a concentration of 5-10 × 105 cpm/ml of
hybridization solution. Duplicate positive clones were rescreened
through one or more cycles until purified plaques were obtained. About
1.3 × 106 independent clones were screened, yielding
two independent, full-length clones, SFD-4A and SFD-21.
Antibody Preparation and Western Blot Analysis--
A peptide
common to the predicted translations of SFD and SFD
(CEMKRSPEEKQEMLQTEGS) and an SFD
-specific peptide
(CKLRGSGVTAETGTUSSSD) were synthesized, coupled to keyhole limpet
hemocyanin, and used for immunization of New Zealand White rabbits (3),
yielding Q48 and Q50 antisera, respectively. For Western blot analysis, protein samples were separated by 11% SDS-PAGE and transferred electrophoretically to nitrocellulose paper. Immunodetection was performed using immune serum at a 1:5,000 dilution and an Amersham ECL
Western blotting system. Immunoblot protocols using rabbit polyclonal
anti-70-kDa subunit antibody (1:5,000 dilution) have been described
previously (3). Polyclonal anti-rat AP50 (µ2) IgY from chicken was
used at a 1:20,000 dilution, and secondary antibody (goat, anti-chicken
IgY) was used at a 1:40,000 dilution. Rabbit polyclonal anti-39-kDa
antiserum was generated against recombinant bovine brain 39-kDa
polypeptide. This antiserum was used at a 1:5,000 dilution. Protein
determination (25) and SDS-PAGE (26) were performed as reported.
Assay of Proton Pumping--
Partially purified proton pump of
clathrin-coated vesicles was reconstituted into liposomes prepared from
pure lipids by the freeze-thaw, cholate dilution method, as reported
(23). Proton pumping was assessed by ATP-dependent
quenching of acridine orange in an Aminco DW 2C dual wavelength
spectrophotometer as A492-540. Proteoliposomes (1.2 µg of protein) were diluted into 1.5 ml of assay
buffer consisting of 150 mM KCl, 2.5 mM
MgCl2, 10 mM Na-Tricine (pH 7.0), and 7 µM acridine orange. Reactions were initiated by the
addition of 1.3 mM ATP and 1 µM valinomycin
and were terminated with the addition of 1 µM
bis-(hexafluoroacetonyl)acetone.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fig. 1 illustrates key preparations used to investigate the possible role of AP50 in function of the clathrin-coated vesicle proton pump. SDS-PAGE (panel A), and immunoblot analysis (panel B) were performed using protein amounts chosen to achieve approximately equal staining of polypeptide(s) in the proximity of 50 kDa. As shown in panel B, AP50 is present in clathrin-coated vesicles (lane 1) but was not detected in partially purified pump after HTP chromatography (lane 2), fully purified pump (lane 3), or SFD (lane 4). As positive controls, the antibody is shown to react with the AP50 component of biochemically prepared µ2 complex (lane 5) and recombinant AP50 (lane 6); the latter has a slightly greater Mr than native AP50 because of histidine residues and a factor Xa cleavage site at the amino terminus.
|
The results of Fig. 1 indicate that AP50 was not present in our pump preparation after HTP chromatography. To further address this point, the distribution of polypeptides eluted during HTP chromatography was examined by SDS-PAGE, as shown in Fig. 2A. Lanes 3-10 show fractions sequentially eluted by a linear (0-300 mM) NaPi gradient, and lane 11 shows a fraction eluted by a 1 M NaPi wash performed after the gradient. Also illustrated are Western blot analysis performed on the fractions of panel A, using antibodies directed against AP50 (panel B), the 70-kDa proton pump subunit (panel C), and the 39-kDa proton pump subunit (panel D). As shown (panel B), AP50 is not eluted by the standard gradient used to prepare proton pump (lanes 3-10) but was released by a 1 M NaPi step wash (lane 11). In contrast, proton pump components are effectively eluted by the 0-300 mM gradient, as indicated by the simultaneous elutions of the 70-kDa subunit of VC (panel C) and the 39-kDa component of VB (panel D) in lanes 3-7. Notably, neither peripheral (70-kDa) nor membrane-associated (39-kDa) components are eluted by the 1 M NaPi wash (panels C and D, lane 11), demonstrating full separation of the pump components and AP50.
|
Next, the HTP elution fraction shown in Fig. 2, lane 5, was subjected to glycerol gradient centrifugation. Resultant purified proton pump (Fig. 1A, lane 3) that is devoid of AP50 (Fig. 1B, lane 3) catalyzes a high rate of proton pumping after reconstitution into liposomes prepared from pure lipids (Fig. 3). Taken together, these data indicate that the 50-kDa component of the V-pump of clathrin-coated vesicles is not AP50, claims to the contrary (13, 14) not withstanding.
|
To determine the genuine molecular identity of the 50- and 57-kDa components of SFD, these polypeptides were purified and subjected to Edman degradation. Both were blocked at their amino termini, and internal peptides were generated by digestion with trypsin or LysC. After separation by reverse phase high performance liquid chromatography, Edman degradation was performed as described under "Experimental Procedures."
The first indication of similarity of the 57- and 50-kDa polypeptides came from the pattern of peptides generated by trypsin digestion. As shown in Fig. 4, nearly identical elution profiles were obtained for the peptides derived from the 57- and 50-kDa polypeptides (panels A and B, respectively). Shown in Table I are the primary sequences obtained from analysis of tryptic and LysC-derived peptides. Notably, sequences obtained from peaks (T2 and T8) of the 57- and 50-kDa polypeptides were identical. Comparison of these sequences with those of data bases revealed that there was no significant sequence homology except for T1, which showed identity with a human expressed sequence tag (TO6491).
|
|
As outlined under "Experimental Procedures," the primary sequences of the peptides prepared from the 50- and 57-kDa polypeptides were used to generate an 856-bp PCR product that was then used to probe a bovine brain cDNA library; ultimately, two independent, full-length clones (SFD4 and SDF 21) were isolated. The sequence of SFD21, its predicted amino acid sequence, and alignment with the peptides of T1, T2, T8, and L22 are shown in Fig. 5. The open reading frame encodes a polypeptide composed of 465 amino acids with a predicted molecular mass of 54,187 Da.
|
Because of the apparent homology found by direct sequencing of the 50- and 57-kDa subunits of SFD, we sought to clone a second cDNA that
might encode the other of the two polypeptides. As outlined under
"Experimental Procedures," a PCR-based approach ultimately yielded
four clones (SFD-RT1, -2, -3, and -4) from bovine brain poly(A)-enriched mRNA. Of these, SFD-RT1, -2, and -3 were identical to one another and to SFD21. SFD-RT4, however, had a sequence identical
to SFD21, except that it contained a 54-bp insert, resulting in a
protein composed of 483 amino acids with a predicted molecular mass of
55,933 Da. The nucleotide sequence of SFD-RT4 is compared with that of
SFD21 in Fig. 5. As shown, these sequences are identical in both the
coding and noncoding regions, save the 54-bp insert at nucleotide
position 727 of SFD21. The predicted amino acid alignments of SFD-RT4,
and SFD21 are of course identical, except for the putative 18-amino
acid insert at position 175 (Fig. 6). Based on the differences in mass of the predicated proteins encoded by
SFD-RT4 and SFD-21, we presumed that these were equivalent to the 57- and 50-kDa components of SFD, respectively. Provisionally, we assume
that these two proteins are isoforms, and we term the product of
SFD-RT4 (and the 57-kDa polypeptide) SFD and the product of SFD-21
(and the 50-kDa polypeptide)
SFD
.3 Data bank searches
revealed that SFD polypeptides have 53% (gene T14F9.1) and
40% (gene F52E1.10) identity to probable homologues in
Caenorhabditis elegans, 23% identity to a probable
homologue (gene SPAC7D4.10) in Schizisaccharomyces
pombe, and 16% identity to a 54-kDa subunit of the vacuolar
proton pump of S. cerevisiae that is encoded by the
VMA13 gene (20); alignments of the predicted amino acid
sequences of these proteins with SFD
and SFD
are shown in Fig.
6.
|
To further establish the relationship of these two polypeptides to SFD,
we generated antibodies to synthetic peptides predicted from the
translated nucleotide sequences of the 57- and 50-kDa components of
SFD. The first of these, Q48, was generated against an epitope common
to SFD and -
, while the second, Q50, was generated against the
predicted unique 18-amino acid insert present in SFD
. Shown in Fig.
7 are the results of Western blot
analysis using these antibodies. As shown, antiserum directed against
the common epitope (Q48) reacts with both the 57- and 50-kDa subunits
present in holoenzyme (panel B, lane 1) and in
purified SFD (panel B, lane 3). In contrast,
antibody Q50, directed against the unique 18-amino acid insert of
SFD
, reacts only with the 57-kDa component of purified pump
(panel C, lane 1) and SFD (panel C,
lane 3). Neither antiserum reacted with V-pump that had been
selectively depleted of SFD (panels B and C,
lane 2).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of this study demonstrate that the clathrin-coated
vesicle proton pump is highly active in the absence of AP50 (Fig. 3)
and that SFD and -
are distinct from AP50. It is important to
note that these conclusions are in part based on the use of the
identical antibody used in the previous reports (13), indicating that
AP50 was required for function of the clathrin-coated vesicle proton
pump. We believe differences between our results and those published
previously are in part due to variations in the methods of preparation
of active H+-ATPase from clathrin-coated vesicles that were
employed in the two studies.
Purification of the clathrin-coated vesicle proton pump, in our hands,
is achieved by an extended protocol consisting of solubilization of
stripped clathrin-coated vesicle membranes with
C12E9, followed by centrifugation at
150,000 × g for 60 min, HTP chromatography, (NH4)2SO4 fractionation, and
glycerol gradient centrifugation. Enzyme, thus prepared, has a specific
activity of 14-16 µmol Pi·mg protein1·min
1 and is dependent upon
phospholipid (optimally, phosphatidylserine) for activation of ATPase
activity, and liposomes for reconstitution of proton pump activity (4).
In contrast, reports of the role of AP50 in the function of
clathrin-coated vesicle proton pump were based on studies of enzyme
prepared by an abbreviated version of our protocol. The isolation
protocol of these studies (13, 14) consisted of treating stripped
vesicles with C12E9, followed by centrifugation
at 100,000 × g, and glycerol gradient centrifugation. The resultant preparation has a specific activity of 6 µmol
Pi·mg protein
1·min
1. It is
thus possible that the preparation of proton pump used by others was
contaminated with AP50 and that their attempts to directly sequence the
50-kDa component yielded residues from the amino terminus of AP50, but
not SFD
, which was not susceptible to amino terminus sequencing in
our hands. Other claims regarding the role of AP50 in pump structure
and function, such as the reported stoichiometry of about 1 mol of
AP50/mol of proton pump (13, 14) remain inexplicable. In particular,
the report that the AP2 complex is itself fundamentally required for
both pump activity and the reassembly of dissociated proton pump
components (14) is in direct conflict with the previous biochemical (6)
and current molecular characterizations of SFD from our laboratory. Irrespective of these points, it is clear that inclusion of HTP chromatography as a pump purification step results in separation of
AP50 from fully functional enzyme (see above); thus, we find no support
for the notion (14) that AP50 is required for acidification catalyzed
by the proton pump of clathrin-coated vesicles.
Molecular cloning of the components of SFD and -
demonstrates
that they differ only by a 54-bp "insert" present in the former. Because of nucleotide identity at all other positions, including noncoding regions at the 3'- and 5'-ends of the clones, it is most
likely that this difference in sequences is due to alternative splicing, as has been shown for a number of V-pump subunits. It is
further possible that SFD
and SFD
are isoforms. Biochemical studies will be required to determine if there are requirements for both components in the function of a single pump or whether a
single form is sufficient for function.
Sequence homology of SFD and -
with the VMA13 product
(20) underscores the apparent similarity in function of these
components in V-pumps from bovine brain and yeast, respectively.
Specifically, SFD was shown to activate Mg-ATPase activity of the
V-pump of clathrin-coated vesicles and to functionally couple ATP
hydrolysis to proton flow. Also, V-pump selectively depleted of SFD
remains intact but cannot support proton pumping (6). These
characteristics are highly similar to those observed in
VMA13 knock-out experiments, where it was found that
the V-pump of yeast vacuoles, although assembled, was inactive with
regard to ATPase and proton pumping activities (20).
Site-specific regulation of V-pump activity is an issue of broad interest that relates to the maintenance and generation of different pH gradients in organelles of the constitutive trafficking pathways and to the role that this interorganelle pH gradient plays in targeting nascent proteins to their ultimate cellular destinations. Although it is possible that AP50 and V-type proton pumps interact to produce some currently undefined function, our data refute the notion that AP50 is an organelle-specific regulator of V-pump function and specifically demonstrate that AP50 is not required for pump activity. We thus are focused on determining the molecular characteristics of SFD and its role in the function of V-type proton pumps.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK-33627.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF041337 and AF041338.
§ To whom correspondence should be addressed: Div. of Molecular Transport, Dept. of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9121. Tel.: 214-648-7606; Fax: 214-648-7542.
1 The abbreviations used are: VB, the bafilomycin-sensitive proton channel of V-type proton pumps; VC, the peripheral, catalytic sector of V-type proton pumps; bp, base pair; C12E9, polyoxyethylene 9-lauryl ether; HTP, hydroxylapatite; PAGE, polyacrylamide gel electrophoresis; SFD (sub-fifty-eight-kDa dimer), the 50-57-kDa polypeptide heterodimer required for function of the vacuolar proton pump of clathrin-coated vesicles; PCR, polymerase chain reaction; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
2 Acidification activity in clathrin-coated vesicle preparations probably arises from Golgi-derived clathrin-coated vesicles, which are associated with AP-1 (17, 18).
3
The predicted masses of SFD and SFD
are in
approximate accord with molecular radii based upon electrophoretic
mobility. At present, we have no evidence of postranslational
modification of either species.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|