Golgi maturation‐dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3

Abstract

Glycosphingolipids are important components of the plasma membrane where they modulate the activities of membrane proteins including signalling receptors. Glycosphingolipid synthesis relies on competing reactions catalysed by Golgi‐resident enzymes during the passage of substrates through the Golgi cisternae. The glycosphingolipid metabolic output is determined by the position and levels of the enzymes within the Golgi stack, but the mechanisms that coordinate the intra‐Golgi localisation of the enzymes are poorly understood. Here, we show that a group of sequentially‐acting enzymes operating at the branchpoint among glycosphingolipid synthetic pathways binds the Golgi‐localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra‐Golgi retro‐transport, acting as a component of the cisternal maturation mechanism. Through these effects, GOLPH3 controls the sub‐Golgi localisation and the lysosomal degradation rate of specific enzymes. Increased GOLPH3 levels, as those observed in tumours, alter glycosphingolipid synthesis and plasma membrane composition thereby promoting mitogenic signalling and cell proliferation. These data have medical implications as they outline a novel oncogenic mechanism of action for GOLPH3 based on glycosphingolipid metabolism.

GOLPH3 and GOLPH3L are broad-spectrum COPI adaptors for sorting into intra-Golgi transport vesicles

“…Logo plot analyses showed that those which bind to GOLPH3/3L have a clear enrichment of basic residues next to the TMD, with blank values dominating further from the TMD, indicating that many of the cytoplasmic tails are not longer than 6-10 residues. Leucine residues are the second most abundant in some positions, consistent with reports that a L-x-x-R/K or L-L-R/K-R/K motifs contribute to binding to GOLPH3 or its yeast ortholog Vps74 (Ali et al, 2012; Tu et al, 2008; Rizzo et al, 2021). We also applied the same analysis to type II proteins that were classified as degraded or non-degraded in the ΔGOLPH3;ΔGOLPH3L cell line, and amongst the degraded set there was a very similar enrichment of membrane-proximal positive residues, again followed by blanks indicative of short cytoplasmic tails.…”

“…1 B). Amongst the interacting Golgi enzymes identified by mass spectrometry, several of the previously reported cargo interactors were identified as hits including GCNT1, EXT1, EXT2, GALNT12, POMGNT1, ST3GAL4 and B4GALT5 (Rizzo et al, 2021; Pereira et al, 2014; Eckert et al, 2014; Isaji et al, 2014; Chang et al, 2013; Ali et al, 2012). Comparing the 73 Golgi-resident membrane proteins enriched by either GOLPH3 or GOLPH3L (P <0.05 compared to GST alone), there was a high degree of overlap (42 common hits, 13 GOLPH3-specific and 18 GOLPH3-specific).…”

“…Knockdown of GOLPH3 in mammalian cells, or the deletion of its ortholog Vps74 in yeast, has been found to cause the mislocalisation of particular Golgi enzymes to the lysosome or vacuole where they are degraded (Tu et al, 2008; Schmitz et al, 2008; Rizzo et al, 2021; Chang et al, 2013). We therefore tested the effect of removing GOLPH3/3L on the stability of two interactors found by affinity chromatography (GALNT7 and GPP130/GOLIM4), and found that the levels of both were greatly reduced in the double knockout background (Fig.…”

“…Once on the membrane, GOLPH3/Vps74 can simultaneously interact with the COPI coat and sample the tails of the enzyme cargo to package them into vesicles recycling from the trans-Golgi to the medial-Golgi (Tu et al, 2008; Schmitz et al, 2008; Eckert et al, 2014). Deletion or depletion of GOLPH3/Vps74 can cause the mislocalisation of its clients to the lysosome or vacuole for degradation (Schmitz et al, 2008; Tu et al, 2008; Rizzo et al, 2021).…”

“…Whilst there seems good evidence that GOLPH3 can direct particular enzymes into COPI vesicles, the scale of its contribution to Golgi enzyme retention is still unclear. For instance, it has been recently proposed that GOLPH3 specifically regulates the retention of enzymes involved in glycosphingolipid synthesis (Rizzo et al, 2021). Moreover, several other roles have been proposed for GOLPH3, including regulating Golgi morphology and forward transport from the trans-Golgi network (TGN), raising the possibility that some of the effects on retention may indirect (Rahajeng et al, 2019; Dippold et al, 2009).…”