Membrane chaperones : protein folding in the ER membrane

Abstract

The plasma membrane (PM), comprised largely of lipids and proteins, is a dynamic structure that establishes the integrity of cells. Newly synthesized PM proteins are initially inserted into the endoplasmic reticulum (ER) prior to being targeted to the PM via the secretory pathway. Many PM proteins are polytopic, i.e., they have multiple transmembrane segments (TMS) and domains located on both sides of the membrane. Polytopic proteins carry out many vital processes, including sensing environmental conditions, and facilitating metabolite transport in and out of cells. The mechanisms that control the functional expression of the PM proteins are not fully understood.

The work documented in this thesis established that four highly specialized accessory proteins within the ER membrane of the yeast Saccharomyces cerevisiae function as membrane chaperones. These chaperones, Shr3, Gsf2, Pho86 and Chs7, are integral components of the ER that are individually required for the functional expression of discrete sets of polytopic PM proteins their substrates. Although these novel chaperones do not share sequence or obvious structural similarity, they function analogously to prevent inappropriate molecular interactions between hydrophobic segments of their substrates as they insert in the ER membrane.

Shr3 plays a critical role in enabling amino acid permeases (AAPs) to fold and attain proper structures required for functional expression at the PM. In the absence of Shr3, AAPs accumulate in the ER, where despite the correct insertion of their twelve TMS, they aggregate forming large molecular weight complexes. Shr3 prevents aggregation and facilitates the functional assembly of independently coexpressed split N- and C-terminal fragments of the general AAP Gap1. Shr3 interacts with the five TMS within the N-terminal fragment and maintains them in a conformation that can post-translationally assemble with the seven TMS in the Cterminal fragment.

AAP aggregates that accumulate in shr3 mutants are redundantly targeted for ERassociated degradation (ERAD) by Doa10 and Hrd1 dependent pathways. In combination, doa10delta hrd1delta mutations stabilize AAP aggregates, and partially suppress amino acid uptake defects of shr3 mutants. Consequently, in cells with impaired ERAD, AAPs are able to attain functional conformations independently of Shr3. These findings illustrate that folding and degradation are tightly coupled processes during membrane protein biogenesis.

A genetic approach identified SSH4, RCR1 and RCR2 as high-copy suppressors of shr3 null mutations. The overexpression of either of these genes increases steadystate AAP levels, whereas their genetic inactivation reduces steady-state AAP levels. Also, suppressor gene overexpression exerts a positive effect on phosphate and uracil uptake systems. Ssh4 and Rcr2 primarily localize to structures associated with the vacuole, however, Rcr2 also localizes to endosome-like vesicles. These findings are consistent with a model in which Ssh4, Rcr2, and presumably Rcr1, function within the endosomal-vacuolar trafficking pathway, where they affect sorting events that determine whether transport proteins are degraded or (re)routed to the plasma membrane.