Differential Stability of Biogenesis Intermediates Reveals a Common Pathway for Aquaporin-1 Topological Maturation

Abstract

Here we re-examine AQP1 biogenesis and show that irrespective of the reporter or fusion site used, oocytes and mammalian cells yielded similar topologic results. AQP1 intermediates containing the first three TM segments generated two distinct cohorts of polypeptides in which TM3 spanned the ER membrane in either an Ncyto/Cexo (mature) or Nexo/Ccyto (immature) topology. Pulse-chase analyses revealed that the immature form was predominant immediately after synthesis but that it was rapidly degraded via the proteasome-mediated endoplasmic reticulum associated degradation (ERAD) pathway with a half-life of less than 25 min in HEK cells. As a result, the mature topology predominated at later time points. We conclude that (i) differential stability of biogenesis intermediates is an important factor for in vivo topological analysis of truncated chimeric proteins and (ii) cotranslational events of AQP1 biogenesis reflect a common AQP1 folding pathway in diverse expression systems.Polytopic membrane proteins are synthesized and oriented in the endoplasmic reticulum (ER) 1 by the ribosome and Sec61 translocon complex (1-3). In the simplest model, topology of each transmembrane (TM) segment is established in a vectoral and sequential manner (N to C termini) (4) as independent signal anchor and stop transfer sequences alternately gate the translocon and the ribosome-translocon junction and direct TM segment integration into the lipid bilayer (cotranslational model) (5-7). However, a growing body of evidence has demonstrated that the final topology of many native proteins is not necessarily established cotranslationally but rather through cooperative interactions between topogenic determinants (TM segments) located within different regions of the polypeptide (post-translational model) (8 -16).One example of the post-translational model occurs during the biogenesis of aquaporin-1 (AQP1), a hydrophobic membrane protein of ϳ29 kDa that exists as a homo-tetramer in cell membranes. AQP1 is a member of the MIP (major intrinsic protein) family (17, 18). In its mature form it exhibits a characteristic topology with six TM segments and two additional short helical regions flanked by conserved NPA motifs that fold inward within the plane of the membrane to form a monomeric, water-selective pore (19,20). AQP1 is expressed in diverse cell types and is localized in the kidney to the proximal tubule and descending limb of the loop of Henle where it plays a major role in renal water reabsorption (18,21).Early biogenesis studies of AQP1 in cell-free systems and microinjected Xenopus oocytes revealed a novel mechanism in which only four of its six transmembrane segments cotranslationally acquired a membrane spanning topology (22). This four-spanning intermediate later matures in the ER membrane to form the final six-spanning structure (23,24). AQP1 biogenesis differs from the cotranslational pathway utilized by a close homolog, AQP4 (25), in part because hydrophilic residues within the N terminus of TM2 (Asn 49 and Lys 51 ) disrupt stop tran...

Control of Translocation through the Sec61 Translocon by Nascent Polypeptide Structure within the Ribosome

“…Of course, this situation would be temporary because residues 56 -58 would again reach Sec61␣ as translation continues, thus giving rise to the biphasic pattern of cross-linking observed. Although the entire AQP1 folding pathway is not yet fully understood, AQP1 biogenesis in the rabbit reticulocyte lysate system has been shown to faithfully reconstitute co-translational integration observed in intact cells (33,51). Our results therefore likely reflect events that occur during native polytopic protein biogenesis and reveal that nascent chain movement during co-translational integration is complex and subject to control by TM-dependent nascent chain folding.…”

“…Second, the decrease in Sec61␣ photocross-linking was not accompanied by a noticeable increase in cross-linking to other translocon components (TRAM or TRAP) (data not shown). Third, AQP1 TM2 is too hydrophilic to terminate translocation and co-translationally span the membrane (31)(32)(33)51), thereby making a transient lateral partitioning into the lipid bilayer highly unlikely.…”

Role of Hsc70 binding cycle in CFTR folding and endoplasmic reticulum–associated degradation

“…For transient transfection, HEK293 cells (3 × 10 5 ) were seeded into 35-mm dishes or six-well plates and, after 24 h, were cotransfected with 1.25 μg of pcDNA3, pc-CBag, or pcI-FLAG-Bag-1 and 1.25 μg of pc-CFTR or pc-CFTR-ΔF508 by calcium-phosphate precipitation (Kingston et al ., 2003). For transfection into COSm6 cells, Trans IT-LT transfection reagent (Mirus, Madison, WI) was used as described (Buck and Skach, 2005). Transfected cells were cultured for 48 h, harvested, flash frozen, and stored at −80°C until immunoblot analysis.…”

The safety dance: biophysics of membrane protein folding and misfolding in a cellular context

“…It is tempting to speculate that this topological heterogeneity may contribute to the poor efficiency with which even wild-type CFTR is known to correctly fold and traffic to the cell surface (Ward & Kopito, 1994). Topological heterogeneity appears to be a feature of the nascent forms of a number of multi-pass α-helical membrane proteins including P-glycoprotein (Pgp, MDR1) (Moss et al, 1998; Skach et al, 1993), sarcoplasmic/endoplasmic reticulum Calcium ATPase 2 (SERCA2) (Bayle et al, 1995), anion exchanger-1 (AE1, band 3) (Kanki et al, 2002), and aquaporin-1 (AQP1) (Buck & Skach, 2005; Lu et al, 2000; Skach et al, 1994). In some cases, the misincorporation of entire TM helices can occur during the biosynthesis of topologically “frustrated” membrane proteins (Gafvelin & von Heijne, 1994), which feature sequences with ambiguous topogenic codes (von Heijne, 2006).…”

“…However, to our knowledge, the mechanisms and molecular players involved in correcting aberrant topomers are currently unclear. Regardless of the mechanism, the reorientation of TM helices can sometimes require hours (Lu et al, 2000) and may often be outpaced by the rapid degradation of misassembled topological intermediates by the proteasome (Buck & Skach, 2005). …”