Abstract
Membrane proteins are sorted to the plasma membrane (PM) via Golgi-dependent trafficking. However, our recent studies challenged the essentiality of Golgi in the biogenesis of specific transporters. Here, we investigate the trafficking mechanisms of membrane proteins by following the localization of the polarized R-SNARE SynA versus the non-polarized transporter UapA, synchronously co-expressed in wild-type or isogenic genetic backgrounds repressible for conventional cargo secretion. In wild-type, the two cargoes dynamically label distinct secretory compartments, highlighted by the finding that, unlike SynA, UapA does not colocalize with the late-Golgi. In line with early partitioning into distinct secretory carriers, the two cargoes collapse in distinct ERES in a sec31ts background. Trafficking via distinct cargo-specific carriers is further supported by showing that repression of proteins essential for conventional cargo secretion does not affect UapA trafficking, while blocking SynA secretion. Overall, this work establishes the existence of distinct, cargo-dependent, trafficking mechanisms, initiating at ERES and being differently dependent on Golgi and SNARE interactions.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
The revised manuscript has addressed all issues raised by the four reviewers and include results from new experiments addressing further the identity of cargo carriers, as asked from three reviewers, using a standard ERES marker, namely Sec16, using our sec31ts system where we can synchronize and better define ER exit of cargoes. These experiments showed rigorously that UapA-GFP and GFP-SynA puncta appearing after transfer of cells into the permissive temperature colocalize significantly with Se16-mCherry (PCC ≈ 0.6), and thus reflect ERES. Given that the puncta marked with UapA or SynA are clearly distinct (Figures 1C,2A, 3A, 5B), this new experiment strongly suggests that there are two distinct ERES, one populated mostly by UapA and the other by SynA. Ideally, to show directly the distinct populations of ERES in the same cell a three-color approach should be used. Indeed, we tried to develop this approach, as proposed by referee 2, by constructing and analyzing strains co-expressing Sec16-BFP or Sec13-BFP as marker of ERES with UapA-GFP and mCherry-SynA. Unfortunately, the BFP affected the function of Sec13 (reduced growth of colonies) and most importantly the blue signal in both cases was extremely low to perform colocalization studies in three channels. Notice also, that overexpression of Sec13 or Sec16, as a tool to increase the blue signal, could not be employed as it leads to growth and secretion defects (our unpublished results). The three-color marker approach can in principle be developed by testing other blue-FP tags (e.g., iRFP), but this extends beyond the scope of the present work, as it would require a long period of time Overall, the new experiments included in the revised manuscript strongly suggest, in line with our other main findings and conclusions, that UapA and SynA are recruited into distinct ERES, and subsequently follow distinct trafficking routes.
Data Availability Statement
Strains and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.
