The Planctomycetes have unique cell architectures with heavily invaginated membrane networks, as here confirmed by three-dimensional models reconstructed from FIB-SEM images of Tuwongella immobilis and Gemmata obscuriglobus. We studied the subcellular proteomes of T. immobilis, and for comparison Escherichia coli, by differential solubilisation followed by LC-MS/MS analysis. More than one thousand proteins were identified in each species. The first Tris-soluble fraction contained mostly cytoplasmic proteins, whereas membrane proteins dominated the second Triton X-100 cell extract. About 50 proteins were exclusive to the third SDS-soluble extract in E. coli, mostly outer membrane and cell surface proteins. A 5-fold higher number of proteins were identified in this fraction in T. immobilis, including predicted cell surface proteins with a prepilin cleavage motif or a Planctomycetes-specific signal peptide. Surprisingly, 50% of the predicted cytoplasmic proteins were exclusively associated with the SDS-soluble protein fraction in T. immobilis. Proteins involved in signal transduction pathway and transcriptional regulation were highly overrepresented in this set, as were also enzymes involved in DNA repair and processing of stable RNAs. Some of these proteins are unique to the Planctomycetes, while others have evolved by gene duplication and domain shuffling. In cases where the paralogs showed different fractionation patterns, it was the most divergent gene copy that was uniquely associated with the SDS-soluble fraction. These results are consistent with the hypothesis that gene duplication and domain shuffling underlie the evolution of new gene functions in the Planctomycetes. We further suggest that repair and recycling of “ageing” molecules play a more important role in the Planctomycetes than in other bacteria due to their large cell sizes, long generation times and life styles.