title: Two-color STED microscopy reveals different degrees of colocalization between hexokinase-I and the three human VDAC isoforms.
abstract: The voltage-dependent anion channel (VDAC, also known as mitochondrial porin) is the major transport channel mediating the transport of metabolites, including ATP, across the mitochondrial outer membrane. Biochemical data demonstrate the binding of the cytosolic protein hexokinase-I to VDAC, facilitating the direct access of hexokinase-I to the transported ATP. In human cells, three hVDAC isoforms have been identified. However, little is known on the distribution of these isoforms within the outer membrane of mitochondria and to what extent they colocalize with hexokinase-I. In this study we show that whereas hVDAC1 and hVDAC2 are localized predominantly within the same distinct domains in the outer membrane, hVDAC3 is mostly uniformly distributed over the surface of the mitochondrion. We used two-color stimulated emission depletion (STED) microscopy enabling a lateral resolution of ~40 nm to determine the detailed sub-mitochondrial distribution of the three hVDAC isoforms and hexokinase-I. Individual hVDAC and hexokinase-I clusters could thus be resolved which were concealed in the confocal images. Quantitative colocalization analysis of two-color STED images demonstrates that within the attained resolution, hexokinase-I and hVDAC3 exhibit a higher degree of colocalization than hexokinase-I with either hVDAC1 or hVDAC2. Furthermore, a substantial fraction of the mitochondria-bound hexokinase-I pool does not colocalize with any of the three hVDAC isoforms, suggesting a more complex interplay of these proteins than previously anticipated. This study demonstrates that two-color STED microscopy in conjunction with quantitative colocalization analysis is a powerful tool to study the complex distribution of membrane proteins in organelles such as mitochondria.PACS: 87.16.Tb, 87.85.Rs.
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany. lkastru@gwdg.de.
authors.forename: Daniel
authors.initials: D
authors.lastname: Neumann
authors.forename: Johanna
authors.initials: J
authors.lastname: Bückers
authors.forename: Lars
authors.initials: L
authors.lastname: Kastrup
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
authors.forename: Stefan
authors.initials: S
authors.lastname: Jakobs