Johana Vallejo

Ketones prevent synaptic dysfunction induced by mitochondrial respiratory complex inhibitors

J. Neurochem. (2010) 114, 130–141.

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. J. Cell. Biochem. 98: 861–871, 2006.

Expression of caveolin-1 in lymphocytes induces caveolae formation and recruitment of phosphofructokinase to the plasma membrane

Compartmentation of carbohydrate metabolism has been shown in a wide range of tissues including reports of one compartment of glycolysis associated with the plasma membrane of cells. However, only in the erythrocyte has the physical basis for plasma membrane‐associated glycolytic pathway been established. We have previously found that phosphofructokinase (PFK) appeared to colocalize with the fairly ubiquitous plasma membrane protein caveolin‐1 (CAV‐1), consistent with a role for CAV‐1 as an anchor for glycolysis to the plasma membrane. To test the hypothesis that CAV‐1 functions as a scaffolding protein for PFK, we transfected human lymphocytes (a cell without CAV‐1 expression) with human CAV‐1 cDNA. We demonstrate that expression of CAV‐1 in lymphocytes results in the formation of caveolae at the plasma membrane and affects the subcellular localization of PFK by recruiting PFK to the plasma membrane. Targeting of PFK by CAV‐1 also was validated by the significant colocalization between the proteins after transfection, which resulted in a correlation of 0.97 ± 0.004 between the two fluorophores. This finding is significant in as much as it illustrates the CAV‐1 feasibility of generating binding sites for glycolytic enzymes on the plasma membrane. We therefore conclude that CAV‐1 functions as a scaffolding protein for PFK and that this may contribute to the elucidation of the basis for carbohydrate compartmentation to the plasma membrane in a wide variety of cell types.

Dissecting the functions of protein-protein interactions: caveolin as a promiscuous partner. Focus on “Caveolin-1 scaffold domain interacts with TRPC1 and IP3R3 to regulate Ca2+ store release-induced Ca2+ entry in endothelial cells”

the centrality of protein complexes and transient protein-protein interactions in cellular physiology is becoming increasingly clear. Although one can readily demonstrate protein-protein interactions with nonphysiological approaches such as immunoprecipitation, it has been more challenging to show