Specific Interactions Mediate Oligomerization and Function of the E. coli Glycerol Facilitator GlpF

Aquaporins, which are involved in human diseases, such as diabetes, skin cancer and brain edema, facilitate the diffusion of water across cellular membranes. In addition, the subfamily of aquaglyceroporines also facilitates the diffusion of glycerol and other linear polyalcohols across cellular membranes. The monomeric E. coli glycerol facilitator GlpF has a quasi two-fold symmetry due to the presence of two genetic repeats. Six transmembrane and two half-membrane-spanning α-helices form the monomeric GlpF channel. GlpF assembles in membranes as higher ordered tetrameric structures. The folding pathway of GlpF has been analyzed by biochemical and biophysical methods. The crucial role of residues W42 and E43 for oligomerization of GlpF has been analyzed in vivo an in vitro. In contrast to W42, E43 is critically involved in oligomerization. While E43 can be replaced by other polar residues, replacement by alanine results not only in a lowered oligomerization tendency but also in a decreased GlpF channel function and in vivo stability. In order to assess a potential role of specific amino acids for folding and stabilization of GlpF, mutants were designed to alter individual transmembrane helix-helix interactions and subsequently the formation of the tertiary and quaternary structure. Additionally, a crucial function of a soluble loop connecting transmembrane helices four and five for assembly and function of GlpF was investigated by site directed mutagenesis and protease cleavage.