Oligomerization of the UapA Purine Transporter Is Critical for ER-Exit, Plasma Membrane Localization and Turnover

Abstract

Central to the process of transmembrane cargo trafficking is the successful folding and exit from the ER (endoplasmic reticulum) through packaging in COPII vesicles. Here, we use the UapA purine transporter of Aspergillus nidulans to investigate the role of cargo oligomerization in membrane trafficking. We show that UapA oligomerizes (at least dimerizes) and that oligomerization persists upon UapA endocytosis and vacuolar sorting. Using a validated bimolecular fluorescence complementation assay, we provide evidence that a UapA oligomerization is associated with ER-exit and turnover, as ER-retained mutants due to either modification of a Tyr-based N-terminal motif or partial misfolding physically associate but do not associate properly. Co-expression of ER-retained mutants with wild-type UapA leads to in trans plasma membrane localization of the former, confirming that oligomerization initiates in the ER. Genetic suppression of an N-terminal mutation in the Tyr motif and mutational analysis suggest that transmembrane α-helix 7 affects the oligomerization interface. Our results reveal that transporter oligomerization is essential for membrane trafficking and turnover and is a common theme in fungi and mammalian cells.

Transporter oligomerization: form and function

“…The high-resolution structure of a thermostabilized form of UapA, trapped in the inward-facing conformation, has revealed further clues about the role that oligomerization has in transport function [11]. UapA was crystallized as a closely associated dimer (Figure 1c,d), confirming the earlier findings [5]. There are many key residues in UapA involved in substrate selectivity [12–14].…”

“…For some membrane proteins, the association of protomers into the correct quaternary arrangement seems to form a key quality control in this trafficking process [10]. This also seems to be true for the H + uric acid–xanthine symporter, UapA, a nucleobase ascorbate transporter (NAT) from Aspergillus nidulans , which requires correct association of individual protomers into a dimer for effective exit from the ER and localization to the plasma membrane [5]. Mutations in TM7 appear to inhibit or impair oligomerization, preventing efficient trafficking to the membrane and increased turnover.…”

“…The most plausible explanation is that the oligomeric arrangement requires the protomers to be correctly folded, with oligomer formation likely to act as a quality control, allowing only proteins in the fully folded state to be trafficked. Interestingly, in the case of UapA, impaired trafficking can be overcome by co-expression of the TM7 mutants with wild-type (WT) UapA [5], suggesting, at least in this context, a dominant positive effect of the WT form.…”

“…The formation of transporter oligomers has been studied by a range of methods, including fluorescence resonance energy transfer (FRET) [1], cross-linking [2], pull-downs [3] and co-immunoprecipitation [4], as well as biophysical characterization using, for example, size-exclusion chromatography–multiangle light scattering (SEC–MALS) [5]. While such approaches have effectively shown the formation of transporter oligomers, more recent investigations using dominant mutants and high-resolution structural studies have started revealing insights into the precise roles of oligomerization for transporter trafficking, function and regulation of function.…”

ER residency of the ceramide phosphoethanolamine synthase SMSr relies on homotypic oligomerization mediated by its SAM domain

“…Together, our data indicate that SAM-mediated oligomerization of SMSr serves a critical role in retaining the enzyme in the ER. This is a remarkable finding in view of the fact that oligomerization has been frequently identified as a prerequisite for efficient export of membrane proteins from the ER4748495051. How oligomerization of SMSr prevents the enzyme from entering COPII vesicles that bud from the ER remains to be established.…”