Endogenous tagging reveals a mid-Golgi localization of the glycosyltransferase-cleaving intramembrane protease SPPL3

TY - JOUR

T1 - Endogenous tagging reveals a mid-Golgi localization of the glycosyltransferase-cleaving intramembrane protease SPPL3

AU - Truberg, Jule

AU - Hobohm, Laura

AU - Jochimsen, Alexander

AU - Desel, Christine

AU - Schweizer, Michaela

AU - Voss, Matthias

N1 - Copyright © 2022. Published by Elsevier B.V.

PY - 2022/11

Y1 - 2022/11

N2 - Numerous Golgi-resident enzymes implicated in glycosylation are regulated by the conserved intramembrane protease SPPL3. SPPL3-catalyzed endoproteolysis separates Golgi enzymes from their membrane anchors, enabling subsequent release from the Golgi and secretion. Experimentally altered SPPL3 expression changes glycosylation patterns, yet the regulation of SPPL3-mediated Golgi enzyme cleavage is not understood and conflicting results regarding the subcellular localization of SPPL3 have been reported. Here, we used precise genome editing to generate isogenic cell lines expressing N- or C-terminally tagged SPPL3 from its endogenous locus. Using these cells, we conducted co-localization analyses of tagged endogenous SPPL3 and Golgi markers under steady-state conditions and upon treatment with drugs disrupting Golgi organization. Our data demonstrate that endogenous SPPL3 is Golgi-resident and found predominantly in the mid-Golgi. We find that endogenous SPPL3 co-localizes with its substrates but similarly with non-substrate type II proteins, demonstrating that in addition to co-localization in the Golgi other substrate-intrinsic properties govern SPPL3-mediated intramembrane proteolysis. Given the prevalence of SPPL3-mediated cleavage among Golgi-resident proteins our results have important implications for the regulation of SPPL3 and its role in the organization of the Golgi glycosylation machinery.

AB - Numerous Golgi-resident enzymes implicated in glycosylation are regulated by the conserved intramembrane protease SPPL3. SPPL3-catalyzed endoproteolysis separates Golgi enzymes from their membrane anchors, enabling subsequent release from the Golgi and secretion. Experimentally altered SPPL3 expression changes glycosylation patterns, yet the regulation of SPPL3-mediated Golgi enzyme cleavage is not understood and conflicting results regarding the subcellular localization of SPPL3 have been reported. Here, we used precise genome editing to generate isogenic cell lines expressing N- or C-terminally tagged SPPL3 from its endogenous locus. Using these cells, we conducted co-localization analyses of tagged endogenous SPPL3 and Golgi markers under steady-state conditions and upon treatment with drugs disrupting Golgi organization. Our data demonstrate that endogenous SPPL3 is Golgi-resident and found predominantly in the mid-Golgi. We find that endogenous SPPL3 co-localizes with its substrates but similarly with non-substrate type II proteins, demonstrating that in addition to co-localization in the Golgi other substrate-intrinsic properties govern SPPL3-mediated intramembrane proteolysis. Given the prevalence of SPPL3-mediated cleavage among Golgi-resident proteins our results have important implications for the regulation of SPPL3 and its role in the organization of the Golgi glycosylation machinery.

U2 - 10.1016/j.bbamcr.2022.119345

DO - 10.1016/j.bbamcr.2022.119345

M3 - SCORING: Journal article

C2 - 36007678

VL - 1869

JO - BBA-MOL CELL RES

JF - BBA-MOL CELL RES

SN - 0167-4889

IS - 11

M1 - 119345

ER -