Transmembrane Topology of α- and β-Subunits of Na+,K+-ATPase Derived from β-Galactosidase Fusion Proteins Expressed in Yeast

Abstract

Various models of the transmembrane topology of the Na ؉ ,K ؉ -ATPase predict either 8 or 10 membrane spans for the ␣-subunit and one to three membrane spans for the ␤-subunit. Structure/function analysis, however, requires precise knowledge about the folding of enzymes. Therefore, the intention of this work was to establish a transmembrane topology model for the subunits of Na LGGYP 236 ) of the dog ␤-subunit. The fusion constructs were expressed in yeast cells for studies on the localization of the fused reporter enzyme. Activity measurements of the reporter enzyme revealed that only intracellular fusion sites lead to active ␤-galactosidase. Indirect immunofluorescence microscopy with cells expressing ␣ 1 /␤-galactosidase and ␤/␤-galactosidase hybrid proteins demonstrated that inactive ␤-galactosidase is associated with the yeast plasma membrane and can be detected from the extracellular side. The data obtained suggest that Pro 236 of the ␤-subunit is located on the extracellular surface, corresponding to a model with one transmembrane segment, and that the ␣-subunit of the Na ؉ ,K ؉ -ATPase consists of 10 membrane-associated spans. They also suggest that a stretch of the ␣ 1 -subunit between membrane spans M7 and M8 might be hidden within the membrane, surrounded by the other hydrophobic spans, in analogy to the P-loop of Na ؉ or K ؉ channels and to the "hourglass" structure of water channels.

Site-directed Chemical Labeling of Extracellular Loops in a Membrane Protein

“…In one study using carboxyl-terminal truncations of the ␣-subunit expressed in yeast (14), the authors place Ala 789 close to the cytoplasmic side and Met 809 in the extracellular loop between M5 and M6. Their results are compared with another 10 membrane-spanning model proposed earlier by Karlish et al (7), who placed Ala 789 close to the extracellular part of M5.…”

“…This is undoubtedly because of the extra mobility and flexibility of the region compared with the amino-terminal region, so that methods that involve disruptive procedures (such as proteolysis and peptide bond cleavage) are prone to generate artifactual observations. We began our investigation in this region by introducing a cysteine at residue 793, which has been predicted to be a part of the M5M6 external loop (7,14,17). Previous studies have shown that the M5M6 hairpin, following proteolysis and removal of K ϩ ions (at 37°C), is released from the membrane (32) to the extracellular space (33).…”

“…Studies on the carboxyl-terminal third of the ␣-subunit have produced conflicting results (7-10, 12-15). Recent data using epitope accessibility claim to establish four transmembrane segments in the carboxyl-terminal region and have a total of eight transmembrane segments (12), whereas several other studies claim to establish that there are 10 transmembrane segments (13)(14)(15).…”

“…These methods are particularly prone to provide misleading information with a protein such as the Na,K-ATPase, where abundant evidence exists demonstrating the mobility or flexibility of its carboxyl-terminal regions. In other methods originally introduced in studies of prokaryotic membrane protein topology, the amino-terminal domain truncations are attached to reporter proteins to determine the topogenic properties of the fusion region (13,14). However, because the complete structure of the protein of interest is not used and functional tests are not then available, it is assumed that the membrane orientation of the truncations faithfully reflects their orientation in the complete proteins.…”

Spectroscopic studies of the interaction of Ca2+‐ATPase‐peptides with dodecyl maltoside and its brominated analog

“…Either four or six C-terminal traverses have been proposed based on hydropathy plots and various experimental approaches (for recent investigations on Na ϩ K ϩ -ATPase see [2,3]). Furthermore, the precise part of the polypeptide chain which is embedded in the membrane is often uncertain (see e.g.…”