Membrane Integration of Na,K-ATPase α-Subunits and β-Subunit Assembly

Abstract

The control of membrane insertion of polytopic proteins is still poorly understood. We carried out in vivo translation/insertion experiments in Xenopus oocytes with combined wild type or mutant membrane segments of the ␣-subunit of the heterodimeric Na,K-ATPase linked to a glycosylation reporter sequence. We confirm that the four N-terminal hydrophobic segments of the ␣-subunit behave as alternating signal anchor/stop transfer motifs necessary for two lipid-inserted membrane pairs. For the six C-terminal membrane segments, however, proper packing depends on specific sequence information and association with the ␤-subunit. M5 is a very inefficient signal anchor sequence due to the presence of prolines and polar amino acids. Its correct membrane insertion is probably mediated by posttranslational hairpin formation with M6, which is favored by a proline pair in the connecting loop. M7 has partial signal anchor function, which may be mediated by the presence of glycine and glutamine residues. The formation of a transmembrane M7/M8 pair requires the association of the ␤-subunit, which induces a conformational change in the connecting extracytoplasmic loop that favors M7/M8 packing. The formation of the M9/M10 pair appears to be predominantly mediated by the efficient stop transfer function of M10. Mutations that provide signal anchor function to M5, M7, and M9 abolish or impede the transport activity of the enzyme. These data illustrate the importance of specific amino acids near or within hydrophobic regions as well as of subunit oligomerization for correct topographical alignment that is necessary for proper folding and/or activity of oligomeric membrane proteins.Many of the important details of the membrane insertion and the maturation of polytopic, oligomeric membrane proteins are still obscure (for reviews, see Refs. 1-3). During the membrane integration, these membrane proteins are subjected to an initial folding process, which is mediated by specific ␣-helical packing (for a review see Ref. 4), interaction with molecular chaperones, and co-translational modifications (for a review, see Ref. 5). Finally, in the case of oligomeric proteins, the maturation process is completed by the assembly of different subunits (for a review, see Ref. 6). Each maturation step is necessary to ultimately result in the correct tertiary structure of oligomeric proteins that is compatible with intracellular transport and function. Analysis of many polytopic membrane proteins is consistent with a model according to which these proteins are produced by the sequential insertion into the endoplasmic reticulum (ER) 1 membrane of signal anchor and stop transfer motifs that generally contain a sequence of about 23 amino acids favoring an ␣-helical conformation and are interrupted by a run of hydrophilic amino acids. The insertion of the first ER-targeting sequence is mediated by the signal recognition particle, which interrupts translation until docked with the signal recognition particle receptor on the ER membrane. Subsequent transmembr...

Endoplasmic Reticulum Quality Control of Oligomeric Membrane Proteins: Topogenic Determinants Involved in the Degradation of the Unassembled Na,K-ATPase α Subunit and in Its Stabilization by β Subunit Assembly

“…On the other hand, M5, M7, and M9 in M1-5, M1-7, and M1-9 ␣-proteins are poor SA sequences, as reflected by the partial glycosylation of these ␣-proteins in an RGS assay. For clarity, the results of these topology studies (Béguin et al, 1998) are summarized in Figure 1. If we assume that the topology studies previously performed with the RGS assay reflect the topology of the molecules devoid of a glycosylation reporter, then the stability of the M1-2, M1-3, and M1-4 ␣-proteins coincides with the efficient SA or ST properties of M1 and M3 or M2 and M4, respectively.…”

“…One of three or four representative experiments is shown. Also summarized at right are results obtained by Béguin et al (1998) on the putative membrane topology of truncated ␣-proteins determined by an RGS assay. Indicated is the percentage of truncated ␣-proteins containing the ectodomain of a ␤ subunit that became glycosylated in an RGS assay performed in Xenopus oocytes in the absence or presence of ␤ subunits.…”

“…The constructs M1-2/5, M1-2/5 P801L/P803L, M1-2/7, M1-2/ 7el, M1-2/7 Q858L/Q869L, M1-2/7el Q858L/Q869L M1-2/7el G860L/G867L, M1-3/ C 5, and M1-3/el/4 were prepared as described (Béguin et al, 1998).…”

“…For this purpose, we generated chimera between the constructs containing N-terminal domains of the ␣ subunit and the 223 amino acids (M81-L303) of the ectodomain of the ␤ 1 subunit of Bufo marinus Na,K-ATPase (Jaisser et al, 1992) containing four glycosylation sites. The chimera were produced as described (Béguin et al, 1998).…”

“…To identify the critical amino acids involved, we tested the degradation of truncated M1-5 ␣-proteins of different length. According to a recently proposed topology model (Moller et al, 1996;Béguin et al, 1998), M5 of the ␣ subunit of Na,KATPase starts at Ser-777, ends at Ile-800, and is connected to M6 by a three-amino-acid-long extracellular loop containing two proline residues (see Table 1). M1-5 ␣-proteins ending at Ile-786 ( Figure 2A, lanes 1 and 2) or at Ala-798 (lanes 3 and 4) were stable during a 48-h chase similar to a M1-4 Q698 ␣-protein ( Figure 1A, lanes 7 and 8), whereas an M1-5 ␣-protein ending at Leu-804 was degraded ( Figure 2A, lanes 5 and 6) similar to the M1-5 G815 ␣-protein ( Figure 1A, lanes 9 and 10).…”