By: 

Ioanna Myronidi, MBW, Stockholm University 

Title: 

Membrane-localized chaperone assisted folding of amino acid transport proteins

Abstract 

The capacity of cells to correctly sense and react to continuously changing external cues is a prerequisite for survival. A multitude of proteins, situated at the outer cell border in the plasma membrane (PM), potentiate cells to perceive environmental conditions and initiate signals to generate appropriate responses. Many of these proteins function as integral components of the PM as receptors, channels or solute transporters. They are typically complex polytopic membrane proteins (PMP) comprised of multiple membrane-spanning segments (MS). In eukaryotic cells, the biosynthesis of PMP takes place at the membrane of the endoplasmic reticulum (ER). Before newly synthesized PMP exit the ER-membrane and are routed to their final membrane destination, through the successive fission and fusion of vesicles of the secretory pathway, they must fold into a three-dimensional structure that ensures their recognition as secretory cargo. PMP folding starts in the ER membrane concomitant with translation as the nascent polypeptide is synthesized and individual MS sequentially partition into the plane of the membrane. However, the final native fold can only be attained when translation completes and all MS are correctly inserted in the ER membrane. To avoid the formation of non-native interactions that can lead to misfolding and aggregation, and to safeguard translation intermediates of PMP from being prematurely degraded, cells employ a number of biogenesis factors localized to the ER membrane. Among those biogenesis factors, highly specialized membrane localized chaperones (MLC) have been identified that are required for the functional expression of discrete sets of related PMP substrates.

Valuable insight into the role of these highly specialized chaperones in PMP biogenesis has been provided in studies of Shr3 (Super High Histidine Resistant), which is a MLC specifically required for the functional expression of the eighteen members of the Amino Acid Permease (AAP) protein family in the yeast Saccharomyces cerevisiae. Shr3 is an integral component of the ER membrane and is comprised of four MS and a cytoplasmically oriented carboxyterminal tail. A mechanistic understanding of how Shr3 facilitates AAP folding is missing, however, it is known that Shr3 exerts its chaperone function primarily via its membrane domain and interacts with COPII coatomer via its cytoplasmic tail. The dual function of Shr3 facilitates folding of newly synthesized AAP in the ER membrane while protecting them from premature ER-associated degradation (ERAD) and promotes inclusion of properly folded AAP into ER-derived COPII-coated secretory vesicles.

The study included in this thesis is focused on the chaperone function coupled to the membrane domain of Shr3. Saturation mutagenesis of this domain has revealed that a low level of sequence specificity is required for establishing productive Shr3-AAP interactions. Further, the results show that luminal loops of Shr3 (L1 and L3) between MS I and II, and III and IV, respectively, affect AAP folding. Mutation induced changes of these loops differentially affect the functional expression of AAP substrates. For example, a deletion of a small portion of luminal loop L3 dramatically affects the ability of Shr3 to interact with and facilitate folding of the general amino acid permease Gap1, but in contrast, has no effect for the AAP homolog Ssy1. We further probe transient Shr3-AAP interactions in vivo with the application of a novel split-ubiquitin approach. We find that Shr3 engages with the first 2 or 4 MS of AAP as they are inserted into the ER. The strength of the interactions progressively increase as more MS are inserted, but weaken as all 12 MS are translated and inserted into the ER membrane. These results are consistent with Shr3 being present and required during the very initial stages of 4 AAP biogenesis. Finally, we report that Shr3 specifically recognizes AAP intermediates and does not interact with intermediates of other unrelated PMP.

Together, the work discussed in this thesis provides a significantly enhanced mechanistic understanding of how Shr3 functions in a co-translational manner as a highly specialized MLC during PMP biogenesis and of the transient MLC-substrate interactions that underlie this process.

You can join the defense via Zoom: https://stockholmuniversity.zoom.us/j/67533298855 

Meeting ID: 675 3329 88 55