Abstract
Kex2p and furin are well-characterized, structurally similar, membrane-bound proteases localized to the trans-Golgi network (TGN) of yeast and animal cells. While their N-terminal and lumenal catalytic domains are highly homologous, they do not share common sorting signals in their C-terminal cytoplasmic tails. To study furin’s localization in yeast, we created a chimera with the N-terminal regions of Kex2p and the C-terminal regions of human furin. Our mating, biochemical, and immunofluorescent studies of the KFp chimeras in yeast have demonstrated sorting consistent with furin’s trafficking patterns in human cells. Next, to see if furin sorting signals are recognized by yeast sorting machinery, key signals were mutated. For two chimeras (KF5p/6p) mating activity was abolished indicating that the Kex2p enzyme is no longer resident to the yeast TGN. This is surprising since this furin sorting signal in animal cells binds a sorting protein (PACS-1) not known to exist in yeast
Highlights
1.1 Kex2p and furin are homologous proteases localized to the trans-Golgi networkConserved cellular machinery is a common feature of eukaryotes
We report our investigations that ask if C-terminal furin sorting signals in yeast are sufficient to localize Kex2p to the trans-Golgi network (TGN)? To study this, we chimerically-fused the N-terminal pre-pro-enzymatic and regulatory domains of the Kex2p coding regions of the yeast KEX2 gene (K) to the C-terminal regions of furin encoded by the human fur cDNA (F) to create chimeric constructs KFJp and KF2p
We initially observed that the chimeric enzyme (KFp) is well-retained in the yeast TGN by several measures, and our findings support a trafficking pattern in yeast that is typical of furin in animal cells. This led us to wonder whether any of the human furin sorting signals are being recognized by conserved sorting machinery native to the yeast cell, and to our surprise we report mating results for site-specific mutations to the acidic cluster (AC) that are consistent with a hypothetical PACS-1-like retrieval of the chimeric enzyme from yeast endosomes to the TGN
Summary
1.1 Kex2p and furin are homologous proteases localized to the trans-Golgi networkConserved cellular machinery is a common feature of eukaryotes. Besides sharing significant structural homology, these two serine proteases are strikingly similar in catalytic specificity and cellular function Both are type I membrane-bound proteins, principally localized to the trans-Golgi network (TGN), that proteolytically process and activate other proproteins passing through the secretory pathway (Fuller et al, 1989b; Takahashi et al, 1995; van den Ouweland et al, 1990). Kex2p cleaves pro-killer toxin, mating factor-α polypeptides 1 and 2, and Kei1p (Julius et al, 1984; Sato et al, 2009; Wickner & Leibowitz, 1976), while over 40 different animal proproteins and animal pathogen proproteins are thought to be cleaved and activated by furin (Thomas, 2002) Both proteases are extensively modified in the endoplasmic reticulum (ER) and Golgi by signal peptidase cleavage, glycosylation, and their own autohydrolysis of the N-terminal pro-region that delivers each as a proteolytically active enzyme to the TGN of their respective cells (Thomas, 2002). The final quarter of each protein (their C-terminal regions) show structural homology, but no significant sequence homology
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