LYSET/TMEM251/GCAF is critical for autophagy and lysosomal function by regulating the mannose-6-phosphate (M6P) pathway

ABSTRACT

Vertebrate cells rely on mannose-6-phosphate (M6P) modifications to deliver most lumenal hydrolases to the lysosome. As a critical trafficking signal for lysosomal enzymes, the M6P biosynthetic pathway has been thoroughly investigated. However, its regulatory mechanism is largely unknown. Here, we summarize three recent studies that independently discovered LYSET/TMEM251/GCAF as a key regulator of the M6P pathway. LYSET/TMEM251 directly interacts with GNPT, the enzyme that catalyzes the transfer of M6P, and is critical for its activity and stability. Deleting LYSET/TMEM251 impairs the GNPT function and M6P modifications. Consequently, lysosomal enzymes are mistargeted for secretion. Defective lysosomes fail to degrade cargoes such as endocytic vesicles and autophagosomes, leading to a newly identified lysosomal storage disease in humans. These discoveries open up a new direction in the regulation of the M6P biosynthetic pathway.

Abbreviations: ER: endoplasmic reticulum; GNPT: GlcNAc-1-phosphotransferase; KO: knockout; LMP: lysosome membrane protein; LYSET: lysosomal enzyme trafficking factor; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; M6P: mannose-6-phosphate; MBTPS1/S1P: membrane-bound transcription factor peptidase, site 1; MPR: mannose-6-phosphate receptor; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TGN: trans-Golgi network.

Macroautophagy/autophagy is a critical clearance and recycling mechanism that targets a range of cargoes, from damaged organelles to harmful protein aggregates. It also participates in the cellular response to starvation by recycling non-essential cellular components. The final destination of autophagy is the lysosome, where over 50 lumenal hydrolases break down various biomaterials and return nutrients to the cytoplasm. Thus, the autophagic flux is critically dependent on the catabolic strength of the lysosomes [1].

While lysosome function requires sufficient synthesis of lumenal hydrolases, their delivery from the ER to the lysosome is also important. In vertebrates, a mannose-6-phosphate (M6P)-dependent sorting system ensures proper delivery of lysosomal hydrolases. After the hydrolases arrive at the cis-Golgi, GNPT/GlcNAc-1-phosphotransferase transfers GlcNAc-1-phosphate to the hydrolases. The GlcNAc component is removed by NAGPA/UCE to expose the M6P modification for recognition by mannose-6-phosphate receptors (MPRs) [2]. MPRs traffic between the trans-Golgi network (TGN), where they bind lysosomal hydrolases, and the endosomal compartments, where they are recycled back to the TGN after releasing the hydrolases [3]. The dissociated hydrolases are then transported to the lysosomes through endomembrane trafficking. Failure in this pathway impairs general lysosomal function and results in severe lysosomal storage diseases. The most common disease-causing mutations in this pathway occur at the genes encoding GNPT subunits, including GNPTAB (encoding α/β) and GNPTG (encoding γ), which lead to mucolipidoses type II and III (MLII and MLIII), respectively [4]. In mucolipidoses patients, lysosome catabolism is greatly reduced, and autophagic turnover is largely blocked.

Because of their importance, components of the M6P biogenesis pathway have been thoroughly characterized. Recently, three independent studies have provided further mechanistic insights into this pathway by discovering LYSET/TMEM251 as a novel regulator of GNPT activity [5–7]. Interestingly, these three studies began with very different biological questions: lysosomal cargo turnover, extracellular nutrient utilization in cancer, and reovirus infection/replication, all of which depend on the digestive strength of the lysosome. Despite their different perspectives, the three studies reached largely consistent conclusions regarding LYSET/TMEM251 function, which will be discussed here.

To identify the genes critical for lysosomal function, Zhang and Yang et al. conducted a genome-wide CRISPR-Cas9 knockout (KO) screen using the degradation of lysosome membrane proteins (LMPs) as a readout [8]. Their top hits included LYSET/TMEM251, a gene of unknown function, and other critical genes in the M6P biogenesis pathway, such as GNPTAB and MBTPS1/site-1 protease. In 2021, LYSET/TMEM251 mutations were reported to cause a novel human disease similar to mucopolysaccharidosis and mucolipidoses type II and III [9]. But the disease mechanism was unclear. To study LYSET/TMEM251 function, these authors deleted the gene and discovered that the degradation of LMPs and endocytosed cargos is severely delayed. In addition, they observed a significant accumulation of SQSTM1/p62 and LC3B-II, indicating a major defect in autophagy. Transmission electron microscopy (TEM) revealed that numerous undigested cargos accumulate in the lysosomes of LYSET/TMEM251 KO cells. All these results indicate a deficiency in the lysosome digestion function. Secretome analysis showed that most lumenal enzymes can no longer reach the lysosome. Instead, they are secreted out of the cell. This trafficking defect is also observed in two other studies [6,7].

Why are lysosomal enzymes hyper-secreted? All three studies discovered that lumenal enzymes lose the M6P modification after deleting LYSET/TMEM251. Using fluorescence imaging and co-IP experiments, they showed that LYSET/TMEM251 is mainly localized to the Golgi and directly interacts with GNPT. Strikingly, mature GNPT disappears in LYSET/TMEM251 KO cells, which explains the M6P defect in lysosomal enzymes. Collectively, all three studies consistently showed that LYSET/TMEM251 is critical for the function of GNPT and M6P biogenesis (Figure 1).

Figure 1.

LYSET/TMEM251 is required for the M6P modification and trafficking of most lysosomal enzymes. Left: In healthy cells, the LYSET/TMEM251-GNPT interaction is required for the M6P modification of lysosomal enzymes at the cis-Golgi. The M6P-modified enzymes are then sorted by MPRs at the TGN. MPRs traffic between the TGN and endosomal compartments to transport lysosomal enzymes. Proper trafficking of lysosomal enzymes is required for all lysosome-dependent functions, such as breaking down endocytosed extracellular proteins, reoviral infection/replication, and digestion of autophagic cargoes. Right: Without LYSET/TMEM251, the GNPT enzyme is either inactive or unstable, which leads to the M6P modification defect and hyper-secretion of most lysosomal enzymes. Under this condition, lysosome-dependent cellular processes are impaired. The green shade indicates that lysosomes are filled with hydrolyses and are functional. The yellow shade indicates that lysosomes lack hydrolyses and are defective.

To explore the physiological relevance of LYSET/TMEM251, Pechincha et al. examined its role in cancer cell growth in vivo [7]. They found that transplanted LYSET/TMEM251-deficient pancreatic cancer cells are strongly impaired in subcutaneous tumor growth. In addition, two animal models have been explored to address the pathology of LYSET/TMEM251 deficiency. In Zebrafish, CRISPR-KO of tmem251 leads to early development failures, including severe edema and skeletal dysplasia, consistent with zebrafish gnptab KO phenotypes. Interestingly, Richards and colleagues established a lyset/tmem251 KO mouse model [6]. The MEF cells derived from the lyset/tmem251 KO mouse show apparent lysosome dysfunction and striking morphological changes under TEM. Blood samples from the KO mice also exhibit high lysosomal enzyme activities, indicating the secretion of lumenal enzymes. However, unlike zebrafish and humans, lyset/tmem251 KO mice do not display obvious clinical symptoms as observed in human patients. These discrepancies between cellular and organismal phenotypes suggest that mice have evolved a compensation mechanism for the loss of lysosomal enzymes. Understanding this compensation mechanism in mice may provide new directions to treat mucolipidoses.

Last, the three studies suggested two alternative models for how LYSET/TMEM251 might regulate GNPT. All three studies observed that activated/mature GNPT disappears after deleting LYSET/TMEM251. Zhang and Yang et al. interpret the data to mean that LYSET/TMEM251 is critical for the MBTPS1/site-1-protease-dependent activation of GNPT and the enzymatic activity after its activation. Accordingly, they named LYSET/TMEM251 as GNPT cleavage and activity factor (GCAF, Figure 2, hypothesis 1). However, they have not experimentally addressed whether mature GNPT might be lost by lysosomal degradation. In contrast, the other two studies proposed that LYSET/Tmem251 is critical for anchoring the activated GNPT at the Golgi. Without LYSET/Tmem251, GNPT/Gnpt activation is normal, but the activated GNPT/Gnpt will quickly mislocalize to the lysosome for degradation. They named the human protein the lysosomal enzyme trafficking factor (LYSET, Figure 2, hypothesis 2). In this model, an obvious question is how defective lysosomes efficiently degrade mislocalized mature GNPT while failing to digest other substrates. Future studies are required to resolve this controversy and answer how the M6P pathway might be regulated through LYSET/TMEM251.

Figure 2.

Two alternative models to explain how LYSET/TMEM251 may regulate GNPT. Left: LYSET/TMEM251 interacts with both MBTPS1/S1P and GNPT, which is required for MBTPS1/S1P-mediated GNPT processing. Without LYSET/TMEM251, the GNPT precursor cannot be cleaved and activated. Right: LYSET/TMEM251 is required for the stability of the activated GNPT. Without LYSET/TMEM251, although the MBTPS1/S1P-mediated GNPT cleavage remains intact, activated GNPT will quickly mislocalize to the lysosome and be degraded.

Funding Statement

This work is supported by the Protein Folding and Diseases Initiative and a MICHR Pathway Pilot grant from the University of Michigan, and NIH grants R01GM133873 and R01HD109346 to ML; National Institute of Child Health and Human Development [R01HD109346; National Institute of General Medical Sciences [R01GM133873].

Disclosure statement

No potential conflict of interest was reported by the authors.