Comigration of Two Autophagosome-Associated Dehydrogenases on Two-Dimensional Polyacrylamide Gels

Abstract

Immunoblotting of two-dimensional polyacrylamide gels (pI 3-10) revealed six cytosolic molecular forms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat hepatocytes. Two of the four full-length (~37 kDa) forms exhibited some binding to sedimentable cellular elements (but not to mitochondria), whereas one full-length and two short (~35 kDa) forms selectively bound to the membranes of autophagosomes and lysosomes. Tryptic fingerprinting by matrix-asssisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the identity of the major full-length forms as GAPDH, but attempts to identify the major short form consistently suggested that this spot represented a different enzyme, 3-a-hydroxysteroid dehydrogenase (3aHSD). Silver staining indicated that this 3aHSD form would selectively bind to autophagosomal and lysosomal membranes. Immunoblotting of more focused 2D gels (pI 6-9) with an antibody raised against 3aHSD demonstrated immunostaining of four 3aHSD forms with masses of about 35 kDa. Autophagosomal membrane preparations were highly and selectively enriched with respect to all of these 3aHSD forms. One of them comigrated with the major short form of GAPDH, accounting for the paradoxical mass spectrometric identification of 3aHSD from this spot. Proteomic analysis by a combination of immunological and mass spectrometric identification methods was thus capable of resolving two comigrating dehydrogenases selectively associated with autophagic organelles.