Chaperoning transmembrane helices in the lipid bilayer

Zhang, Qi; Ye, Yihong

doi:10.1083/jcb.202012041

“…Considering the low pH within the vacuole, our data do not support Ypq1 to be an exporter of positively charged amino acids (Jezegou et al, 2012); rather, our experiments imply that Ypq1 functions as an importer. Strikingly, purified Ypq1 depends on lysine (or arginine) for its stability in vitro, consistent with our in vivo observation that lysine withdrawal can trigger the ubiquitination and degradation of Ypq1 (Arines et al, 2021;Li et al, 2015b;Zhang and Ye, 2021).…”

Section: Introductionsupporting

confidence: 89%

Lysosome transporter purification and reconstitution identifies Ypq1 pH-gated lysine transport and regulation

Arines

¹

,

Wielenga

²

,

Burata

³

et al. 2023

Preprint

0

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Lysosomes achieve their function through numerous transporters that import or export nutrients across their membrane. However, technical challenges in membrane protein overexpression, purification, and reconstitution hinder our understanding of lysosome transporter function. Here, we developed a platform to overexpress and purify the putative lysine transporter Ypq1 using a constitutive overexpression system in protease- and ubiquitination-deficient yeast vacuoles. Using this method, we purified and reconstituted Ypq1 into proteoliposomes and showed lysine transport function, supporting its role as a basic amino acid transporter on the vacuole membrane. We also found that the absence of lysine destabilizes purified Ypq1 and causes it to aggregate, consistent with its propensity to be downregulated in vivo upon lysine starvation. Our approach may be useful for the biochemical characterization of many transporters and membrane proteins to understand organellar transport and regulation.

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“…A genome-wide CRISPR/Cas9 screen reveals essential components for lysosome function Previously, we and others have identified a conserved ubiquitin-and ESCRT-dependent pathway that controls the turnover of lysosome membrane proteins (LMP) [11][12][13][14][15][16][17][18][19][20][21] . In a recent study, we discovered two fast-degrading human LMPs, RNF152 and LAPTM4A, that are constitutively ubiquitinated and internalized into the lysosome lumen by the ESCRT machinery for degradation 17 .…”

Section: Resultsmentioning

confidence: 99%

GCAF(TMEM251) regulates lysosome biogenesis by activating the mannose-6-phosphate pathway

Zhang

¹

,

Yang

²

,

Li

³

et al. 2022

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The mannose-6-phosphate (M6P) biosynthetic pathway for lysosome biogenesis has been studied for decades and is considered a well-understood topic. However, whether this pathway is regulated remains an open question. In a genome-wide CRISPR/Cas9 knockout screen, we discover TMEM251 as the first regulator of the M6P modification. Deleting TMEM251 causes mistargeting of most lysosomal enzymes due to their loss of M6P modification and accumulation of numerous undigested materials. We further demonstrate that TMEM251 localizes to the Golgi and is required for the cleavage and activity of GNPT, the enzyme that catalyzes M6P modification. In zebrafish, TMEM251 deletion leads to severe developmental defects including heart edema and skeletal dysplasia, which phenocopies Mucolipidosis Type II. Our discovery provides a mechanism for the newly discovered human disease caused by TMEM251 mutations. We name TMEM251 as GNPTAB cleavage and activity factor (GCAF) and its related disease as Mucolipidosis Type V.

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Lysosomal membrane transporter purification and reconstitution for functional studies

Arines,

Wielenga,

Henn

et al. 2024

MBoC

0

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Lysosomes achieve their function through numerous transporters that import or export nutrients across their membrane. However, technical challenges in membrane protein overexpression, purification, and reconstitution hinder our understanding of lysosome transporter function. Here, we developed a platform to overexpress and purify the putative lysine transporter Ypq1 using a constitutive overexpression system in protease- and ubiquitination-deficient yeast vacuoles. Using this method, we purified and reconstituted Ypq1 into proteoliposomes and showed lysine transport function, supporting its role as a basic amino acid transporter on the vacuole membrane. We also found that the absence of lysine destabilizes purified Ypq1 and causes it to aggregate, consistent with its propensity to be downregulated in vivo upon lysine starvation. Our approach may be useful for the biochemical characterization of many transporters and membrane proteins to understand organellar transport and regulation.

show abstract

Chaperoning transmembrane helices in the lipid bilayer

Cited by 3 publications

References 10 publications

Lysosome transporter purification and reconstitution identifies Ypq1 pH-gated lysine transport and regulation

Lysosome transporter purification and reconstitution identifies Ypq1 pH-gated lysine transport and regulation

GCAF(TMEM251) regulates lysosome biogenesis by activating the mannose-6-phosphate pathway

Lysosomal membrane transporter purification and reconstitution for functional studies

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