Comigration of two autophagosome-associated dehydrogenases on two-dimensional polyacrylamide gels

Immunoblotting of two-dimensional polyacrylamide gels (pl 3-10) revealed six cytosolic molecular forms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat hepatocytes. Two of the four full-length (similar to 37 kDa) forms exhibited some binding to sedimentable cellular elements (but not to mitochondria), whereas one full-length and two short (similar to 35 kDa) forms selectively bound to the membranes of autophagosomes and lysosomes. Tryptic fingerprinting by matrix-assisted 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-alpha-hydroxysteroid dehydrogenase (3 alpha HSD). Silver staining indicated that this 3 alpha HSD form selectively bound to autophagosomal and lysosomal membranes. Immunoblotting of more focused 2D gels (pl 6-9) with an antibody raised against 3 alpha HSD demonstrated immunostaining of four 3 alpha HSD forms with masses of about 35 kDa. Autophagosomal membrane preparations were highly and selectively enriched with respect to all of these 3 alpha HSD forms. One of them comigrated with the major short form of GAPDH, accounting for the paradoxical mass spectrometric identification of 3 alpha HSD from this spot. Proteomic analysis by a combination of immunological and mass spectrometric identification methods was thus capable of resolving two comigrating dehydrogenoses selectively associated with autophagic organelles.