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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 21, 2005 as doi:10.1096/fj.04-2380fje. |
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Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, Missouri, USA
1Correspondence: Department of Medical Pharmacology and Physiology, MA 415 Medical Sciences Building, University of Missouri, Columbia, MO 65212, USA. E-mail: HardinC{at}missouri.edu
SPECIFIC AIMS
Although glycolytic enzyme localization to the plasma membrane has been proposed to underlie glycolytic compartmentation in a wide range of cells, only in the erythrocyte has the mechanistic basis for such enzyme localization been understood. The aim of this study is to determine whether CAV-1 acts as a scaffolding protein for targeting of endogenous phosphofructokinase in lymphocytes (a highly glycolytic cell type normally devoid of CAV-1).
PRINCIPAL FINDINGS
1. Transfection of lymphocytes with CAV-1 cDNA results in expression of CAV-1 protein and caveolae formation at the plasma membrane
To test whether a new association of CAV-1 and PFK could occur, we took advantage of the fact that lymphocytes are highly glycolytic cells that lack expression of caveolin proteins. We transfected lymphocytes with CAV-1 cDNA and confirmed the consequent expression of CAV-1 protein by Western blot analysis. We further analyzed whether the transfected protein was functionally expressed at the plasma membrane by characterization of the formation of caveolae invaginations as evidenced by transmission electron microscopy (Fig. 1
). These results demonstrate the role of CAV-1 in caveolae formation and validate other reports in which CAV-1 expression resulted in de novo formation of caveolae in lymphocytes.
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2. Expression of CAV-1 results in significant recruitment of PFK to the plasma membrane
To test the hypothesis that endogenous PFK and transfected CAV-1 can interact and are colocalized at the plasma membrane, confocal microscopy was used to determine the distribution and colocalization of PFK and CAV-1 after CAV-1 cDNA transfection into lymphocytes. We observed a significant expression of immunoreactive CAV-1 after transfection compared with control, validating the results obtained by Western blot analysis. We also observed a consequent marked shift in the distribution of PFK with decreased immunoreactivity of PFK to subcellular regions and increased immunoreactivity at the plasma membrane as compared with control. Distribution of PFK throughout the entire cell was analyzed by creating an intensity profile from the central Z plane of each image. The pseudo-color scale in the intensity profile from each image demonstrated a uniform distribution of PFK in wild-type lymphocytes (Fig. 2
). However, we found a redistribution of PFK after CAV-1 transfection with increased PFK intensity to the periphery of transfected lymphocytes and decreased PFK intensity at subcellular locations as compared with control cells (Fig. 2)
. These results indicate that CAV-1 induces recruitment of PFK to the plasma membrane and validates the role for CAV-1 as a scaffolding protein for PFK.
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CONCLUSIONS AND SIGNIFICANCE
In this study we demonstrate that caveolin-1 acts as a strong scaffolding protein for targeting of endogenous phosphofructokinase in lymphocytes. This finding is significant inasmuch as it illustrates the CAV-1 feasibility of generating binding sites for glycolytic enzymes on the plasma membrane.
Confocal microscopy demonstrated a uniform distribution of PFK in wild-type lymphocytes. Lymphocytes are highly glycolytic cells with a dense and dynamic actin cytoskeleton. Since the PFK distribu-tion appears rather uniform in wild-type lymphocytes, either PFK is not localized to the cell ultrastructure or PFK is localized to a variety of densely packed ultrastructures throughout the cytoplasm. Considerable evidence suggests that glycolytic enzymes are found in association with the microtubules and F-actin. Therefore, one might speculate that the apparent uniform expression of PFK in wild-type lymphocytes is a result of the association of PFK with the dense cytoskeleton.
Expression of CAV-1 into lymphocytes resulted in redistribution of this subcellular localization of PFK with significant recruitment of PFK to the plasma membrane and significant colocalization between the proteins. These results might indicate that the affinity of PFK for CAV-1 is much stronger than the affinity provided by the actin cytoskeleton and microtubules or perhaps that PFK is not bound to any particular subcellular components in lymphocytes. Regardless of the particular localization of PFK, expression of CAV-1 resulted in substantial PFK redistribution with most of PFK targeted to the plasma membrane. The demonstration that CAV-1 functions as a scaffolding protein for the membrane recruitment of PFK is a key step in elucidating the physical basis for glycolytic membrane interactions and their role in the organization of carbohydrate metabolism as observed in our previous studies of VSM. Consideration of these data in the physiological context of cellular organization provides new insight into the ways in which the glycolytic enzymes may be compartmentalized within mammalian cells.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2380fje; doi: 10.1096/fj.04-2380fje
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