(Received for publication, August 1, 1995; and in revised form, January 24, 1996)
From the
We recently identified a 28-kDa protein in the intestinal brush
border that resembled tropomyosin in terms of size, homology, and
helical content. This protein contained 27 heptad repeats, nearly all
of which began with leucine, leading to its name zipper protein.
Subsequent analysis, however, indicated that both a 49-kDa and a 28-kDa
immunoreactive protein existed in intestinal brush-border extracts.
Using 5`-rapid amplification of cDNA ends analysis, we extended the
N-terminal sequence of zipper protein to the apparent translation start
site. This additional sequence contained a putative transmembrane
domain and two potential tryptic cleavage sites C-terminal to the
transmembrane domain which would release a 28-kDa cytoplasmic protein
if utilized. The additional sequence was highly homologous to members
of the B-G protein family, a family with no known function.
Immunoelectron microscopy showed that zipper protein was confined to
the membrane of the microvillus where it was in close association with
brush-border myosin 1 (BBM1). Recombinant zipper protein (28-kDa
cytoplasmic portion) blocked the binding of actin to BBM1 and inhibited
actin-stimulated BBM1 ATPase activity. In contrast, zipper protein had
no effect on endogenous or K/EDTA-stimulated BBM1 ATPase activity.
Furthermore, zipper protein displaced tropomyosin from binding to
actin, suggesting that these homologous proteins bind to the same sites
on the actin molecule. We conclude that zipper protein is a
transmembrane protein of the B-G family localized to the intestinal
epithelial cell microvillus. The extended cytoplasmic tail either in
the intact molecule or after tryptic cleavage may participate in
regulating the binding and, thus, activation of BBM1 by actin in a
manner similar to tropomyosin.
The intestinal brush border contains a highly cell-specific form
of myosin 1 called brush-border myosin 1
(BBM1)()(1) . BBM1 is part of an ever increasing
family of non-muscle myosins (2, 3, 4, 5, 6) which are
characterized by homology to conventional muscle myosins in terms of
their ATP and actin binding domains in the N-terminal head of the
molecule, but lack the
helical rod-like tail domain which permits
conventional myosins (i.e. myosin 2) to dimerize. Like other
myosins, BBM1 has actin-stimulated ATPase activity and, in the presence
of ATP and calcium, generates movement along actin
filaments(7, 8) . BBM1 is capable of binding both to
the actin core and to the membrane of the
microvillus(7, 9, 10, 11) . Binding
to the membrane alters its activity by as yet uncertain
mechanisms(7, 12) . Binding to the membrane appears to
involve the C-terminal portion of the molecule and requires acidic
phospholipids(13) . This ability to bind both actin and
membranes suggests that BBM1 plays a role in membrane
transport(14) . Regulation of the binding of BBM1 to the
membrane or to actin is not thoroughly understood. Tropomyosin blocks
binding of BBM1 to actin(8) , but tropomyosin is not found in
the microvilli where BBM1 is located(11) . We (15) recently described a protein with structural homology to
tropomyosin which at the light microscopic level colocalized with BBM1
to the brush border. Because of its extended leucine zipper motif we
call this protein zipper protein. Subsequent to the initial report, we
have determined that the originally reported structure is the
cytoplasmic tail of a larger protein in the B-G protein family, a
family of proteins with no known function. The data in this report
indicate that zipper protein is a transmembrane protein of the
intestinal microvillus whose cytoplasmic tail regulates the binding of
BBM1 to actin.
Figure 1: The nucleotide and amino acid sequence of zipper protein.
The samples for zipper protein
localization were cryoprotected in 2.3 M sucrose in 0.05 M phosphate buffer, pH 7.2, for 30 min, then frozen in liquid
nitrogen and sectioned using a Leica Ultracut E ultramicrotome,
equipped with an FC4E cryoattachment at -100
°C(19, 20) . The sections were collected on nickel
grids and coated with Formvar and carbon. The sections were first
incubated with 50 mM NHCl in PBS for 15 min.
Nonspecific binding was blocked by incubation with 5% newborn calf
serum (Life Technologies, Inc.) in 10 mM Tris, pH 7.6,
containing 500 mM NaCl (TBS) for 30 min. The grids were then
incubated with anti-zipper protein antiserum(1092) or with preimmune
serum, diluted 1:25 in TBS containing 5% fetal bovine serum for 1 h,
followed by washing for 30 min. Binding of the antibody was revealed
with goat anti-rabbit IgG, conjugated to 15-mm colloidal gold (Zymed).
The grids were washed and stained with 0.3% uranyl acetate in 2%
methylcellulose.
BBM1 was detected on pyridoxal phosphate-fixed
tissues, embedded into Lowicryl HM23 resin, polymerized by UV at
-25 °C. The sections were first incubated with 50 mM NHCl in PBS for 15 min. Nonspecific binding was
blocked by incubation for 30 min with 1% bovine serum albumin, 0.1%
cold water fish skin gelatin (Sigma), 0.0001% Tween 20 in 10 mM Tris, pH 7.6, containing 500 mM NaCl (blocking buffer).
The grids were then incubated with affinity-purified anti-BBM1
antibody, diluted in blocking buffer for 1 h, followed by washing with
blocking buffer for 30 min. Binding of the antibody was detected as for
zipper protein. The grids were washed and stained sequentially with 2%
osmium tetroxide in PBS, 2% uranyl acetate, and 1% lead citrate.
All sections were examined and photographed in a Zeiss 10A transmission electron microscope operated at 60 kV.
Subsequent to our initial report(15) , we became
aware that our original clones encoding zipper protein may have
contained ``introns'' which interfered with the determination
of the beginning of the open reading frame in the 5` end of the
sequence, a frequent occurrence in clones of B-G
proteins(22, 23) . ()Therefore, we repeated
the sequencing of the 5` end of zipper protein, using mRNA from gut
mucosa (rather than a cDNA library) as starting material, and 5`-RACE
technology. Nine clones were fully sequenced in both directions.
Although varying in length at the 5` end, all clones encoded the same
protein. The consensus sequence when coupled to the previously
determined sequence of zipper protein encoded a protein of 49,272
daltons (374 residues) (Fig. 1). This sequence showed a high
degree of homology with the 5` end of the B-G proteins obtained from an
erythroid cell library published by Miller et al.(22) as shown in Fig. 2.
Figure 2: Homology of zipper protein and four B-G proteins cloned and sequenced by Miller et al.(22) from an erythroid cell library. The homology is most striking in the N-terminal half of the proteins which includes the transmembrane domain (amino acids 159-178 of zipper protein).
The hydropathy plot of the N-terminal region of zipper protein (Fig. 3) indicates a 20-amino acid stretch of hydrophobic amino acids (amino acids 159-178) which is highly conserved among the B-G proteins; as seen in Fig. 2, conservative amino acid substitutions are the only differences in the 7 positions not completely homologous among these proteins in this region with the exception of a proline in D39371 instead of an alanine in the other proteins. This likely represents a transmembrane domain. Immediately C-terminal to this putative transmembrane domain is a highly hydrophilic domain (amino acids 179-194) which contains 7 basic residues in pairs or triplets. Proteolytic cleavage in this basic region would release a 28-kDa protein. This region also is highly homologous with the B-G proteins. The homology among the B-G proteins in the remainder of the structure is less well preserved, although all proteins demonstrate the extended leucine zipper motif. The leucine zipper motif suggests that these proteins exist as coiled coils in their C-terminal cytoplasmic domains. Comparing the migration of the C-terminal portion of zipper protein on denaturing versus nondenaturing gels, we found the expected size (28 kDa) on denaturing gels but an apparently much larger protein (approximately 120 kDa) on nondenaturing gels suggesting multimer formation and/or aberrant migration of an asymmetric molecule (data not shown).
Figure 3: Hydropathy plot of zipper protein. Hydrophobicity is indicated by positive values. Charged residues are indicated by a + or - as appropriate. The boxed region includes the hydrophobic region from amino acids 159-178, the likely transmembrane domain, and a hydrophilic region from amino acids 179-194 which contains a number of basic residues in doublets or triplets providing a likely site for proteolytic cleavage.
To determine whether zipper protein existed as the full-length or cleaved product in the intestinal epithelium, we examined extracts of intestinal mucosa by Western analysis using antibodies raised to different regions of the deduced sequence. The results are shown in Fig. 4in which the same blot is probed with both antibodies. The antibody(1470) raised to a region near the tail of the sequence (amino acids 351-363) detected two proteins, one with the expected size for the cytoplasmic domain of zipper protein (28 kDa) and one substantially larger (49-50 kDa). A second antibody(1468) raised to the basic region adjacent to the putative transmembrane domain (see below) (amino acids 187-200) recognized only the 49-kDa band. Both antibodies recognized only the 49-kDa species in purified brush-border membranes (data not shown). The structure for zipper protein shown in Fig. 1has a calculated molecular mass of 49.3 kDa, suggesting that it is the complete sequence. The smaller band (28 kDa) detected by antibody 1470 is consistent with cleavage in the polybasic region of zipper protein C-terminal of the transmembrane domain and in a region that would disrupt the epitope recognized by antibody 1468.
Figure 4: Western analysis of intestinal extracts was performed using two antibodies. 1470 was raised to an epitope in the cytoplasmic domain, whereas 1468 was raised to an epitope spanning the putative tryptic cleavage region near the transmembrane domain. The same blot was probed with both antibodies. Each antibody sees the same 49-kDa protein, but 1470 sees a 28-kDa protein as well which we postulate is the cytoplasmic tail of the 49-kDa protein. In this gel, the molecular marker for 49.5 kDa ran slightly below the 49-kDa protein.
To determine more precisely the location of zipper protein within the brush border, we performed immunogold localization at the electron microscopic level and compared the results to the immunolocalization of BBM1. The results are shown in Fig. 5. The left panel shows the immunolocalization of zipper protein; the right panel shows the immunolocalization of BBM1. Although BBM1 could be detected in embedded tissue, zipper protein localization at the ultrastructural level required cryosections. The zipper protein was found exclusively in the microvilli of the intestinal epithelial cells where it appeared closely associated with the membrane. No labeling of the basolateral membrane or cytoplasmic organelles was seen. Goblet cells and cells in the submucosa did not express detectable levels of zipper protein. BBM1 was also found exclusively in the microvilli. However, BBM1 had both a membrane-associated and intravesicular localization. As for zipper protein, BBM1 was not found in the intermicrovillar domain of the apical plasma membrane or invaginations of the apical plasma membrane, basolateral membrane, or other subcellular organelles. When the anti-zipper protein or anti-BBM1 antiserum was replaced with preimmune serum, no gold particles were seen over the brush border.
Figure 5: Immunoelectron microscopic localization of zipper protein and BBM1. The left panel shows the immunolocalization of zipper protein; the right panel shows the immunolocalization of BBM1. The gold particles mark the presence of zipper protein exclusively in the membrane, whereas BBM1 is found within the microvillus as well. Two different methods were required for these two different proteins as described under ``Experimental Procedures.'' Preabsorption of the antisera eliminated all background staining.
We then assessed the amounts of zipper protein in purified brush-border membranes in comparison with actin and myosin 1. In purified brush-border membranes, zipper protein existed only in the 49-kDa form at a concentration of 0.4 ng/µg of protein, whereas in intestinal extracts its concentration was considerably higher (2.5 ng/µg of protein) and both 49-kDa and 28-kDa forms were present. These data suggest that the cleaved product of zipper protein is lost during purification of the membranes. Actin and myosin were found in purified membranes at a concentration of 47 and 56 ng/µg of protein, respectively.
The structure of the presumed cytoplasmic portion of zipper protein resembles tropomyosin, raising the possibility that it functions like tropomyosin in regulating myosin/actin interactions. To test this possibility, we evaluated first the ability of zipper protein to block the binding of actin to BBM1, a property previously found for tropomyosin(8) . The results are shown in Fig. 6. The labeling of this figure reflects the fact that one-half of the original incubate was used for the binding analysis (i.e. 0.5 µg of BBM1, 1 µg of actin, and 0.1 to 3 µg of zipper protein). The first three panels (A, B, and C) depict the same blot of supernatant samples probed for BBM1, actin, and zipper protein, respectively. The final three panels (D, E, and F) depict the same blot of pellet samples probed for BBM1, actin, and zipper protein, respectively. The graphs indicate the percent of the total BBM1 and actin in the supernatants and pellets, respectively. In the absence of zipper protein and ATP, all BBM1 was precipitated with actin (A and D). As zipper protein was increased from 0.1 to 3 µg, an increasing amount of BBM1 (up to 30%) was displaced from the actin precipitate. Maximal inhibition occurred at 1 µg of zipper protein or approximately a 2:1 molar ratio with actin. ATP alone released 70% of the BBM1 from the actin pellet, and zipper protein potentiated this effect of ATP (to 100% release at 3 µg). Actin (B and E) remained in the precipitate in the absence of ATP despite increasing concentrations of zipper protein. In the presence of ATP, 18% of the actin was found in the supernatant; zipper protein from 0.1 to 0.3 µg increased actin solubilization to 40%, but at higher concentrations inhibited this solubilization such that, at 3 µg of zipper protein, all actin was found in the precipitate regardless of ATP concentration. Zipper protein (C and F) remained primarily in the supernatant where it showed the expected increase with increasing addition of zipper protein.
Figure 6: Zipper protein inhibition of actin binding to BBM1. BBM1 and actin were incubated with increasing amounts of zipper protein. The amount of BBM1 (actin and zipper protein) displaced from actin cosedimentation was quantitated immunologically by absorption unto PVDF membranes of the proteins in the supernatant following centrifugation of the actin. The pellets were analyzed in similar fashion after SDS-PAGE and blotting onto the same type of membrane. The same blot for the supernatants (A, B, and C) and the equivalent blot for the pellets (D, E, and F) were probed for BBM1 (A and D), actin (B and E), and zipper protein (C and F). The graphs of the data for BBM1 and actin show the percent distribution of each sample between supernatant and pellet. The doublet seen on Western analysis of BBM1 represents intact BBM1 (upper band) and its degradation product (lower band). The densitometric data shown in the graph included both bands. The graphs for zipper protein show the densitometry data. For A-C, the first five lanes show the displacement of BBM1 from actin with increasing amounts of zipper protein. The two lanes on the right are controls with BBM1 alone (labeled 0 actin, 0 zp) or BBM1 plus actin which was not centrifuged after incubation (labeled Total). The incubations on the top row were performed in the absence of ATP, whereas the incubations on the bottom row were performed in the presence of 5 mM ATP. For D-F, the labeling of the lanes is as for A-C, but with the samples incubated in the presence of ATP being found to the right of those incubated in its absence.
We repeated this approach to determine whether zipper protein and tropomyosin share the same binding site on actin. The results are shown in Fig. 7. In panel A of this figure, increasing amounts of zipper protein (from 0 to 3 µg) were added to 1 µg of actin prior to incubation with 0.3 µg of tropomyosin. In the absence of zipper protein, essentially all the tropomyosin was pelleted with the actin. In the presence of increasing amounts of zipper protein, less tropomyosin was pelleted with actin, such that displacement was nearly complete at the highest concentration of zipper protein (3 µg). In panel B, the experiment shown tested the ability of tropomyosin to displace zipper protein in its binding to actin. In the absence of tropomyosin, 64% of the zipper protein (0.3 µg) was removed from the supernatant by 1 µg of actin. Coincubation with 0.3 µg of tropomyosin released all the actin-bound zipper protein into the supernatant. In a separate experiment, we found that maximum binding of zipper protein to 1 µg of actin occurred at 1-3 µg of zipper protein indicating saturation of the actin at a molar ratio of approximately 2:1 zipper protein:actin (data not shown).
Figure 7: Zipper protein inhibition of tropomyosin binding to BBM1. Tropomyosin and actin were incubated with increasing amounts of zipper protein. The amount of tropomyosin (A) or zipper protein (B) displaced from actin cosedimentation was quantitated immunologically by absorption unto PVDF membranes of the proteins in the supernatant following sedimentation of the actin. In the case of tropomyosin (A), the results are calculated as a percent of the total amount of tropomyosin in the incubate, whereas the actual densitometry results for zipper protein (B) are shown.
The ability of zipper protein to inhibit actin-stimulated (A) but not K/EDTA-stimulated (B) BBM1 ATPase is shown in Fig. 8. In this experiment, 10 µg of actin was used to stimulate the ATPase activity of 1 µg of BBM1. This concentration of actin stimulated endogenous BBM1 ATPase activity by 2.17-fold. Increasing the zipper protein from 1 to 10 µg eliminated completely the actin stimulation with no further inhibition at 30 µg. Zipper protein had no effect on BBM1 ATPase activity in the absence of actin. As for actin binding (Fig. 6), zipper protein had its maximal effect at a 2:1 molar ratio with actin. In contrast to its inhibition of actin-stimulated BBM1 ATPase, zipper protein had no effect on K/EDTA-stimulated ATPase (B).
Figure 8: Zipper protein regulation of BBM1 ATPase activity. B shows the ability of zipper protein to inhibit actin-stimulated ATPase activity without altering the endogenous ATPase activity of BBM1. A shows that zipper protein has little effect on K/EDTA-stimulated BBM1 ATPase activity. The error bars enclose mean and range of duplicates. Data points without error bars had duplicate determinations within the span of the symbol.
In our original report of zipper protein(15) , we identified 3 clones which differed primarily at their 5` ends but contained an essentially identical open reading frame encoding a 28-kDa protein which we called zipper protein. Although the nucleotide sequences were homologous to the family of proteins called B-G proteins, proteins originally purified from chicken erythrocytes but found also in a variety of immune tissues(24) , the lack of hybridization of the zipper protein cDNAs to marrow and spleen cells indicated to us that we were dealing with a different protein(15) . Subsequent to our original report, we became aware of the potential inclusion of introns into the cDNAs from which zipper protein was cloned (22, 23) which could account for the variation in the 5` end and mislead us in terms of identifying the full open reading frame. Therefore, we repeated the sequencing of zipper protein using mRNA directly from the chick intestinal mucosa. Nine clones were sequenced and all encoded the same protein. The additional N-terminal sequence had a much higher homology with the equivalent region of the B-G proteins sequenced by Miller et al.(22) which were cloned from a library prepared from erythroid cells of a 14-day-old chick embryo. Furthermore, Western analysis of intestinal mucosal extracts demonstrated the existence of a 49-kDa protein indicating that the predicted B-G-like protein was actually produced. Thus zipper protein is clearly a member of the B-G family.
B-G proteins are characterized by a highly conserved N-terminal domain with a variable C-terminal domain(22, 23, 25) . Zipper protein fits this description in that it is highly homologous to the N-terminal portion of the erythroid B-G proteins but not to the C-terminal region. B-G proteins appear to be integral membrane proteins oriented with the N-terminal region extracellular and with a single transmembrane domain. The putative transmembrane domain of zipper protein is highly homologous to that in the erythroid B-G proteins (Fig. 2). The extracellular domain of B-G proteins is not N-glycosylated and shows homology with members of the immunoglobulin superfamily(22) . B-G proteins are encoded by a polygenic region of the chick major histocompatibility locus (26) and were originally thought to be limited in distribution to erythroid tissues. With their demonstration in thrombocytes and lymphoid tissue (24) , a potential role for B-G proteins in immunologic function has been proposed. However, B-G proteins have also been described in the intestine(27) , and an understanding of their role remains elusive.
We (15) were impressed with the high degree of homology between the cytoplasmic portion of zipper protein and tropomyosin and proposed that zipper protein might function as the tropomyosin of the microvillus in terms of regulating BBM1/actin interactions(8, 15) . Tropomyosin, although present in the intestinal epithelial cell, is not present in the microvillus(28) . Zipper protein, on the other hand, is found exclusively in the microvillus. Our immunolocalization studies indicate that zipper protein is inserted into the microvillus membrane, consistent with its possession of a transmembrane domain. However, Western analysis indicates that the cytoplasmic portion can be cleaved from its anchor in the membrane. During preparation of purified brush-border membranes, all of the cleaved product is lost, such that the measured levels of zipper protein in the purified brush-border membrane preparation are considerably below those found in crude intestinal extracts. The highly basic region adjacent to the transmembrane domain is a likely target for proteolytic cleavage and, if utilized, would release the 28-kDa protein found on Western analysis. The antibody that spans the putative cleavage site(1468) did not detect the 28-kDa band, only the intact 49-kDa protein, providing additional evidence that this region is the site of cleavage. Although protease inhibitors were employed during the extraction of the intestinal mucosa for Western analysis, we cannot be certain that the proteolysis occurred in vivo. In either case, the cytoplasmic portion of zipper protein does have tropomyosin-like properties in that it blocks the binding of actin to BBM1 and tropomyosin and inhibits actin-stimulated BBM1 ATPase activity. A 2:1 molar ratio of zipper protein to actin appears to be maximally effective and is sufficient to saturate binding of zipper protein to actin. This ratio is greater than that seen for tropomyosin which saturates actin binding at a molar ratio of approximately 1:3.5 tropomyosin:actin(29) . This would imply that zipper protein has a lower affinity for actin than does tropomyosin or binds to a different site on the actin molecule. The latter possibility seems unlikely in view of the ability of zipper protein to displace tropomyosin from actin and vice versa. At this point, it is not clear whether zipper protein can perform these tropomyosin-like functions while anchored to the membrane or whether the cytoplasmic portion needs to be released from its membrane anchor to be effective. Our measurements of zipper protein levels in purified brush-border membranes relative to actin and myosin indicate that the concentration of intact zipper protein is far below that required to saturate actin suggesting that its regulatory role may be limited. Conceivably, the intact molecule acts as a receptor for some as yet undefined signal leading to release of the intracellular domain which acts locally to regulate actin/BBM1 interactions. Whether other B-G proteins will have similar functions remains for future investigation. But the concept that B-G proteins can control membrane movement by regulating the activity of the submembrane cytoskeleton is intriguing.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U49098[GenBank].