The ER membrane chaperone Shr3 co-translationally assists biogenesis of related plasma membrane transport proteins

Proteins with multiple membrane-spanning segments (MS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. In Saccharomyces cerevisiae, Shr3 is an ER membrane-localized chaperone (MLC) that is specifically required for the functional expression of amino acid permeases (AAP), a family of eighteen transporters comprised of 12 MS. Here, comprehensive scanning mutagenesis and deletion analysis of Shr3, combined with a modified split-ubiquitin approach, were used to probe chaperone-substrate (Shr3-AAP) interactions in vivo. A surprisingly low level of sequence specificity in Shr3 underlies Shr3-AAP interactions, which initiate early as the first 2 MS of AAP partition into the membrane. The Shr3-AAP interactions successively strengthen and then weaken as all 12 MS partition into the membrane. Thus, Shr3 acts transiently in a co-translational manner to prevent MS of AAP translation intermediates from engaging in non-productive interactions, effectively preventing AAP misfolding during biogenesis.

Expression of Genes Related to Sugar and Amino Acid Transport and Cytokinin Metabolism during Leaf Development and Senescence in Pisum sativum L.

Gene editing is becoming the plant breeding tool of choice, but prior to targeting a gene for editing, a knowledge of the gene family members (GFMs) controlling yield is required in the specific crop plant. Critical to yield are components from senescing leaves. We targeted genes controlling senescence in Pisum sativum and the release and transport of carbohydrates and amino acids from the source leaves to the pods and seeds. The expression of GFMs for cytokinin biosynthesis (IPT) and destruction (CKX), sucrose transporters (SUT), Sugar Will Eventually be Exported Transporters (SWEET), amino acid permeases (AAP), and cell wall invertases, was determined using RT-qPCR. GFMs were differentially expressed in leaves of different ages. The expression of many gene family members was lower in the expanding sink leaves compared with the senescing leaves, with the exception of two PsAAP GFMs and PsCKX5, which were highly expressed. Expression of specific PsSWEETs, SUTs, and AAPs increased in the mature and/or senescing leaves. Expression of PsIPTs was least in the mature source leaves, but as strong in the senescing leaves as in the young source leaves. PsCKX7 was expressed in source and senescing leaves. We discuss the potential impact of the targeted reduction of specific PsCKX GFMs on source-sink relationships.

Transition of yeast Can1 transporter to the inward-facing state unveils an α-arrestin target sequence promoting its ubiquitylation and endocytosis

Substrate-transport–elicited endocytosis is a common control mechanism of membrane transporters avoiding excess uptake of external compounds, though poorly understood at the molecular level. In yeast, endocytosis of transporters is triggered by their ubiquitylation mediated by the Rsp5 ubiquitin-ligase, recruited by α-arrestin–family adaptors. We here report that transport-elicited ubiquitylation of the arginine transporter Can1 is promoted by transition to an inward-facing state. This conformational change unveils a region of the N-terminal cytosolic tail targeted by the Art1 α-arrestin, which is activated via the TORC1 kinase complex upon arginine uptake. Can1 mutants altered in the arginine-binding site or a cytosolic tripeptide sequence permanently expose the α-arrestin–targeted region so that Art1 activation via TORC1 is sufficient to trigger their endocytosis. We also provide evidence that substrate-transport elicited endocytosis of other amino acid permeases similarly involves unmasking of a cytosolic Art1-target region coupled to activation of Art1 via TORC1. Our results unravel a mechanism likely involved in regulation of many other transporters by their own substrates. They also support the emerging view that transporter ubiquitylation relies on combinatorial interaction rules such that α-arrestins, stimulated via signaling cascades or in their basal state, recognize transporter regions permanently facing the cytosol or unveiled during transport.