Analysis of glycolytic enzyme co-localization in Drosophila flight muscle
1 Department of Biology, Syracuse University, Syracuse, NY 13224,
USA
2 Department of Genetics and Molecular Biology, Cornell University, Ithaca,
NY 14853, USA
* Author for correspondence (e-mail: dtsulliv{at}syr.edu)
Accepted 13 March 2003
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Summary |
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Key words: glycolytic enzyme, co-localization, Drosophila, flight muscle, myofibril, proteinprotein interaction, M-line, Z-disc, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerol mutase
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Introduction |
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Several years ago, we attempted to bring the power of genetic analysis, both classical and molecular, to extend our understanding of the functional significance of co-localized enzymes of a pathway. We initiated studies using the well-characterized glycolytic pathway in Drosophila and we chose adult flight musculature as a tissue for study. Muscle has well defined cytoarchitecture and, more importantly, offers the possibility of analyzing the functional importance of enzyme interactions using the selectable phenotype flight as a marker of genetic perturbation.
Studies were begun using the glycolytic enzyme glycerol-3-phosphate
dehydrogenase (GPDH). This enzyme and its gene (gpdh) have been well
characterized (Kotarski et al.,
1983; MacIntyre and Davis,
1987
; von Kalm et al.,
1989
). A large number of mutant alleles at this locus have been
isolated and some result in the loss of flight ability. When flight muscle was
analyzed using immunofluorescence microscopy, GPDH was found localized at
Z-discs and M-lines. When immune serum was used to react with sections of
thoraces of GPDH null mutants that are known not to synthesize any
immunologically cross-reactive material, no specific labeling of myofibrils
was detected. Localization of two other glycolytic enzymes,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase, was also
detected in flight muscle at Z-discs and M-lines, in a manner
indistinguishable from that of GPDH. The striking result was that in GPDH null
mutants, GAPDH and aldolase are not properly localized at Z-discs or M-lines
but are found instead in the extramyofibrillar spaces
(Wojtas et al., 1997
).
When molecular characterization of the gpdh gene and its
expression was completed (von Kalm et al.,
1989), it was determined that this gene encodes three isoforms
produced through differential splicing of three exons at its 3' end. One
of these, GPDH-1, is found only in thoracic musculature and is the only
isoform found in this tissue. The tissue specificity of this isoform offered a
unique opportunity to generate genetically engineered flies that could make
the other isoforms but not GPDH-1. Significantly, GPDH-1, as compared with the
other two isoforms, has a C-terminal tripeptide, Q-N-L. This tripeptide is
encoded by a specific exon, exon 8, that is separated from the rest of the
gene by a 1-kb intron.
We then set out to determine if GPDH-1 localization is accomplished, at
least in part, through its C-terminal tripeptide. Transgenic flies that
produce only GPDH-3 were made (Wojtas et
al., 1997). This isoform is distinguished from GPDH-1 by lacking
the C-terminal tripeptide and is not normally produced in muscle. In these
transgenic flies, the endogenous gpdh promoter was used to drive
GPDH-3 production, and the resident gpdh locus was homozygous for
alleles unable to produce GPDH. We found that in flight muscle of these
engineered flies, GPDH-3 is not localized at Z-discs and M-lines but is found
in the extramyofibrillar spaces. As is the case in GPDH null mutants, GAPDH
and aldolase are also not localized at Z-discs and M-lines in these engineered
flies.
Once we established that the glycolytic enzymes are localized in an interdependent manner, we next wished to determine if this localization has functional significance. We found that genetically engineered flies that produce only GPDH-3 in their flight muscles cannot fly normally even though they have appreciable GPDH activity in their flight muscles. Therefore, the interdependent co-localization of glycolytic enzymes, and not simply their activity in flight muscle cells, is required for flight.
A long-term goal is to identify proteinprotein interactions that support glycolytic enzyme localization. In this paper, we present the analysis of an expanded set of glycolytic enzymes and more precisely examine details of glycolytic enzyme localization at M-lines and Z-discs to determine if enzyme binding at the two sites has different properties. We have begun to identify some of the pairwise proteinprotein interactions that may be involved in myofibrillar glycolytic enzyme co-localization.
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Materials and methods |
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Myofibril preparation
Myofibrils were prepared from adults of either sex that were 26 days
post emergence. Flies were immobilized by chilling, and heads and abdomens
were separated from the thorax. Thoraces were opened in fixative (see below)
and dissected free of non-muscle material; fixation was for 60 min on ice. The
fixative comprised 4% paraformaldehyde, 0.5% glutaraldehyde, 0.1 mol
l-1 phosphate buffer, pH 7.2. Following fixation, the thoraces were
homogenized, and myofibrils were collected on Transwell polyester membrane
filters (12 mm diameter, 3.0 µm pore size; Corning) and then washed with
three changes of 0.5 mol l-1 phosphate, 0.15 mol l-1
NaCl, 0.1 mol l-1 Tris. The filters were incubated with primary
antibodies for 1 h, washed with three changes of phosphate buffer saline (PBS)
containing 0.1 mol l-1 Tris, pH 7.2. The filters were then
incubated with non-immune goat serum (Pierce) as a blocking agent for 15 min
and then incubated with fluorescein isothiocyanate (FITC)-labeled goat
anti-rabbit serum. The filters were washed three times in PBS-Tris. The
filters were cut out from their frame and mounted on a slide for fluorescence
microscopy.
Antibodies
Antibodies against Drosophila GPDH and GAPDH were generated by
immunizing rabbits with proteins purified from flies as previously described
(Skuse and Sullivan, 1985;
Sullivan et al., 1985
).
Antibodies against Drosophila aldolase (Ald), triose phosphate
isomerase (TPI), phosphoglycerate kinase (PGK) and phosphoglycerol mutase
(PGLYM) were generated by cloning full-length cDNA coding for each protein
into the vector pTrcHis (Invitrogen). The fusion protein was isolated and the
sample sent to Josman Laboratories, CA, USA for immunization of rabbits and
antisera preparation. Each serum was used at the maximal dilution possible to
obtain an adequate signal; in each case, the concentration was less than or
equal to a 1:1000 dilution.
Visualization of actin
Actin was visualized by staining with rhodamine-conjugated phalloidin.
Myofibrils were mounted in glycerol and viewed using a 100x 1.3 NA
objective mounted on a Nikon TE-300 microscope fitted for epifluorescence.
Excitation and emission of rhodamine and FITC were accomplished with Texas red
and FITC optimized filter sets (Nikon), respectively. Images were captured
with a MicroMax 5 MHz CCD camera (Roper/Princeton Instruments).
Molecular biology
Molecular biological techniques were performed using standard procedures
(Maniatis et al., 1982). The
yeast two-hybrid system was used as described
(Bai and Elledge, 1997
) with
the plasmids pAct II and Pas-I. Yeast cells containing the desired pair of
plasmids were generated by co-transformation and selection for
leu+,trp+ cells. Interaction was tested by selecting for
leu+,trp+,his+ cells in media supplemented
with 25 mmol l-1 3-amino triazole to assess activation of the
his promoter. Spot tests were then done to verify activation of the
ß-gal promoter. Cells demonstrating transcription from both
his and ß-gal promoters were grown in liquid culture
and assayed fluorimetrically for ß-galactosidase using the FluorAce
reporter assay kit (BioRad).
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Results |
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In isolated myofibrils, M-lines and Z-discs show a pattern of GPDH fluorescence labeling similar to that of sectioned thoraces, but it often appears that M-lines and Z-discs have an alternating pattern of bands with different relative intensities (Fig. 1). This pattern is sometimes seen in sectioned material but the difference between alternating bands is not as striking. In the isolated myofibrils shown in Fig. 1, it is apparent that the brighter band appears to be interrupted centrally, i.e. the band appears as a doublet. We have extended our observations on using isolated myofibrils to determine whether other glycolytic enzymes are similarly localized to those previously studied and to determine whether the variable M-lineZ-disc intensity pattern might provide a suggestion as to the mechanisms of glycolytic enzyme localization in the myofibril.
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A similar pattern of localization of GAPDH and aldolase at M-lines and Z-discs is also apparent in isolated myofibrils (Fig. 2); the same is true for TPI, PGK and PGLYM (Fig. 3). Each enzyme has a similar pattern of localization, i.e. alternating bright and less-intense bands. The brighter band appears to be bisected by a narrow line of reduced or no antibody binding. Using antisera against PGK, we find the same alternating bands with different fluorescent intensity. However, the brighter band does not show the central dark zone. We do not know whether this represents a real difference in PGK localization or is due to technical issues. We have found in all our studies, both immunofluorescence microscopy and western blotting, that levels of anti-PGK-reactive material in flight muscle are much lower than for the other glycolytic enzymes we have studied. In addition, we sometimes find when using the other antisera that the central dark band is not seen in sarcomeres that appear weakly fluorescent, possibly due to inadequate fixation. However, the central point is that six glycolytic enzymes that catalyze consecutive reactions along the glycolytic pathway are found similarly localized in flight muscles at M-lines and Z-discs.
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To determine whether M-lines or Z-discs are represented by the more brightly fluorescing bands, we conducted a double label experiment using FITC-labeled anti-GPDH and rhodamine-labeled phalloidin. Results of these experiments are shown in Fig. 4. Phalloidin binds to actin and its staining pattern shows labeling along the A-band, as expected. There is an alternating pattern of darker phalloidin-unlabeled bands. We interpret the narrow bands as representing Z-discs and the broader bands as representing M-lines. Phalloidin binds to actin and, since actin does not cross the area of the M-line but does penetrate into the Z-disc, our expectation is that the actin fluorescent pattern will show broader M-lines and narrower Z-discs and be therefore distinguishable. Comparing the GPDH pattern to the phalloidin pattern reveals that the bright GPDH bands correspond to the broader non-phalloidin-labeled M-lines, and the less intensely anti-GPDH-labeled bands are the Z-discs.
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We have also found that localization of the glycolytic enzymes at M-lines is more labile that at Z-discs. In developing procedures for the preparation of myofibrils, it became apparent that rapid fixation is required to retain the maximal M-lineZ-disc labeling pattern. We have compared the intensity of fluorescence in muscles dissected in fixative with that found in muscles dissected in buffers, incubated and then transferred to fixative. The fluorescence intensity at M-lines can be lost or much reduced, as shown in Figs 5, 6, if muscles are not dissected directly in fixative. Thoracic muscles were dissected in contraction buffer (100 mmol l-1 KCl, 5 mmol l-1 KPO4, 4 mmol l-1 CaCl2, 5 mmol l-1 MgCl2, 5 mmol l-1 ATP, pH 7.0) and incubated on ice in contraction buffer for 5 min (Fig. 5) or for 20 min (Fig. 6A). They were then fixed and examined for GPDH localization. The fluorescent pattern is greatly diminished at M-lines and in some cases disappears completely. The Z-disc fluorescence is minimally affected. Comparison of Fig. 6A with Fig. 6B (myofibrils simultaneously labeled with phalloidin) confirms that it is the M-line that loses anti-GPDH labeling. In contraction buffer, M-lines appear narrower, as would be expected in muscles put under contraction conditions. The loss of M-line localization also occurs in relaxation buffer (data not shown), showing that it is the delay in fixation, not the act of contraction, that results in the loss of glycolytic enzymes from the M-line. Therefore, during preparation, the signal from M-lines, which is more intense than from Z-discs, in rapidly fixed myofibrils, is more easily perturbed than the signal from Z-discs. This implies that the organization or binding of glycolytic enzymes at M-lines and Z-discs is different and that glycolytic enzymes are bound less tightly at M-lines than at Z-discs.
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We have reported (Wojtas et al.,
1997), using sections of flight muscles, that sarcomeric
localization of GAPDH and aldolase does not occur in the absence of proper
GPDH localization. This is the case using GPDH null mutants or in genetically
engineered flies that ectopically produce only the GPDH-3 isoform in muscle
cells.
Similar results are obtained using preparations of myofibrils. Myofibrils prepared from GPDH null mutants stained with anti-GPDH show no immunoreactive material when stained with anti-GPDH, confirming the specificity of the immunofluorescence microscopy (Fig. 7). Neither GAPDH or aldolase (Fig.·7) nor TPI, PGK or PGLYM (Fig. 8) are found localized at M-lines or Z-discs in myofibrils isolated from GPDH null mutants. Therefore, all glycolytic enzymes that have been analyzed to date behave as a set and are similarly and interdependently localized.
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We have initiated several biochemical and genetic approaches to identify
those proteinprotein interactions that are functionally important for
glycolytic enzyme co-localization. These would include interactions that occur
among members of the set of glycolytic enzymes and interactions between the
glycolytic enzymes and other muscle structural proteins. Initial results on
interactions among the glycolytic enzymes have been obtained using the yeast
two-hybrid assay system. cDNAs for each of the glycolytic enzymes PFK,
aldolase, GPDH, GAPDH, TPI, PGK and PGLYM were introduced into the
vectors pAS1 and pACT2 and then transformed into yeast cells of both a and
mating types. Yeast matings were conducted in all pairwise
combinations where each of the parental plasmids contained a different member
of the set of seven glycolytic enzymes. Cells of the genotype
his+,leu+,trp+ were selected and tested for
ß-galactosidase expression. Those testing positive for expression from
both his and ß-gal promoters were then grown in liquid
culture, and ß-galactosidase activity was measured fluorimetrically.
Whereas most pairs of glycolytic enzymes failed to show evidence of
interaction, two glycolytic pairs GPDH/GAPDH and GPDH/PGLYM
did show activation of both promoters, suggesting that these proteins can
interact in yeast cells. The cells containing these plasmid pairs and the
activities of several control plasmid combinations in terms of units of
ß-galactosidase activity are reported in
Table 1.
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Subunits of GPDH-1 and GPDH-3 do interact, as might be expected, since GPDH is an active dimer. Subunit interaction is also seen in the Ald/Ald combination, also expected since aldolase has a homotetrameric structure. However, we see no evidence of aldolase interactions with either GPDH-1 or with GAPDH. We also fail to see evidence of any interactions when using TPI, PGK, PGLYM or PFK in any pairwise combination with any other members of this set of glycolytic enzymes. Of note is the interaction of GPDH-3 and GAPDH. If the interactions between GPDH and GAPDH seen using the yeast two-hybrid assay are reflective of an in vivo functionally relevant interaction, this suggests that these glycolytic interactions may occur in cells other than muscle cells, where GPDH-3 is not found.
We have attempted to identify possible targets of glycolytic binding in muscles by testing the set of glycolytic enzymes with fusion proteins of the muscle proteins. Proteins tested include flight muscle actin, actinin, troponin C and troponin I. In no cross did results establish an interaction of one of these proteins with a glycolytic enzyme. Since the yeast two-hybrid system seems most well suited to detect fairly strong proteinprotein interactions, these results suggest that if interaction between any of these proteins does occur, it is likely to be weak. This conclusion also applies to the negative tests for interaction among members of the set of glycolytic enzymes.
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Discussion |
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The function of glycolysis in muscle is to provide ATP for myosin ATPase to
enable contraction. This may be either directly from the phosphorylation
reactions of the glycolytic pathway or by providing pyruvate for mitochondrial
oxidative phosphorylation. Glycolysis presumably also plays a role, either
directly or indirectly, in providing ATP for myosin phosphorylation. Since
flight ability is greatly diminished when the glycolytic enzymes are not
co-localized (Wojtas et al.,
1997), a rather direct role of glycolytic enzyme co-localization
in muscle activity is suggested. But the glycolytic enzymes, at least for the
most part, are not localized at the A-band, the sites of myosin localization
and ATP utilization. At present, we have insufficient understanding of the
mechanisms that connect glycolytic enzyme co-localization and muscle
contraction.
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Acknowledgments |
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References |
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Bai, C. and Elledge, S. (1997). The yeast two-hybrid system. In The Yeast Two-Hybrid System (ed. P. Bartel and S. Fields), pp. 11-28. New York: Oxford University Press.
Kotarski, M. A., Pickert, S., Leonard, D. A., LaRosa, G. J. and
MacIntyre, R. J. (1983). The characterization of
-glycerophosphate dehydrogenase mutants in Drosophila
melanogaster. Genetics 105,387
-407.
MacIntyre, R. and Davis, M. (1987). A genetic
and molecular analysis of -glycerolphosphate cycle in Drosophila
melanogaster. In Isozymes: Current Topics in Biological
Research, vol. 14 (ed. M. Ratazzi, J.
Scandalios and G. Whitt), pp. 195-224. New York: Alan
R. Liss.
Maniatis, T., Fritsch, D. F. and J. Sambrook. (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Ovadi, J. and Srere, P. (2000). Macromolecular compartmentation and channeling. In Inter. Nat. Review of Cytology, vol. 192, pp.255 -280: Academic Press.
Skuse, G. R. and Sullivan, D. T. (1985). Developmentally regulated alternate modes of expression of the Gpdh locus of Drosophila. EMBO J. 4,2275 -2280.[Abstract]
Srere, P. A. (1987). Complexes of sequential metabolic enzymes. In Annual Review of Biochemistry, vol. 56 (ed. P. Boyer, I. Dawid and A. Meister), pp.89 -124. Palo Alto: Ann. Rev. Inc.[CrossRef][Medline]
Sullivan, D. T., Carroll, W. T., Kanik-Ennulat, C. L., Hitti, Y., Lovett, J. A. and VonKalm, L. (1985). Glyceraldehyde-3-phosphate dehydrogenase from Drosophila melanogaster: identification of two isozymes forms encoded by separate genes. J. Biol. Chem. 260,4345 -4350.[Abstract]
von Kalm, L., Weaver, J., DeMarco, J., MacIntyre, R. J. and
Sullivan, D. T. (1989). Structural characterization of the
-glycerol-3-phosphate dehydrogenase-encoding gene of Drosophila
melanogaster. Proc. Natl. Acad. Sci. USA
86,5020
-5024.[Abstract]
Wojtas, K., Slepecky, N., vonKalm, L. and Sullivan, D. (1997). Flight muscle function in Drosophila requires co-localization of glycolytic enzymes. Mol. Biol. Cell 8,1665 -1675.[Abstract]
Wu, X. M., Gutfreund, H. and Chock, P. B. (1992). Kinetic method for differentiating mechanisms for ligand exchange reactions application to test for substrate channeling in glycolysis. Biochemistry 31,2123 -2128.[Medline]
Wu, X. M., Gutfreund, H., Lakatos, S. and Chock, P. B. (1991). Substrate channeling in glycolysis: a phantom phenomenon. Proc. Natl. Acad. Sci. USA 88,497 -501.[Abstract]