Overexpression of caveolin-1 results in increased plasma membrane targeting of glycolytic enzymes: the structural basis for a membrane associated metabolic compartment.
Although membrane-associated glycolysis has been observed in a variety of cell types, the mechanism of localization of glycolytic enzymes to the plasma membrane is not known. We hypothesized that caveolin-1 (CAV-1) serves as a scaffolding protein for glycolytic enzymes and may play a role in the organization of cell metabolism. To test this hypothesis, we over-expressed CAV-1 in cultured A7r5 (rat aorta vascular smooth muscle; VSM) cells. Confocal immunofluorescence microscopy was used to study the distribution of phosphofructokinase (PFK) and CAV-1 in the transfected cells. Areas of interest (AOI) were analyzed in a central Z-plane across the cell transversing the perinuclear region. To quantify any shift in PFK localization resulting from CAV-1 over-expression, we calculated a periphery to center (PC) index by taking the average of the two outer AOIs from each membrane region and dividing by the central one or two AOIs. We found the PC index to be 1.92 +/- 0.57 (mean +/- SEM, N = 8) for transfected cells and 0.59 +/- 0.05 (mean +/- SEM, N = 11) for control cells. Colocalization analysis demonstrated that the percentage of PFK associated with CAV-1 increased in transfected cells compared to control cells. The localization of aldolase (ALD) was also shifted towards the plasma membrane (and colocalized with PFK) in CAV-1 over-expressing cells. These results demonstrate that CAV-1 creates binding sites for PFK and ALD that may be of higher affinity than those binding sites localized in the cytoplasm. We conclude that CAV-1 functions as a scaffolding protein for PFK, ALD and perhaps other glycolytic enzymes, either through direct interaction or accessory proteins, thus contributing to compartmented metabolism in vascular smooth muscle.
Raikar
Leena S
LS
Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, Missouri 65212, USA.
Vallejo
Johana
J
Lloyd
Pamela G
PG
Hardin
Christopher D
CD
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism*; D051242:Caveolin 1 / Q000096:biosynthesis* / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology*; D002478:Cells, Cultured; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism; D015870:Gene Expression; D006019:Glycolysis / Q000502:physiology*; D006801:Humans; D018613:Microscopy, Confocal; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000378:metabolism; D051381:Rats
D051242:Caveolin 1; D010732:Phosphofructokinase-1; C068652:1-phosphofructokinase; D005634:Fructose-Bisphosphate Aldolase