Selective retrograde transport from endosomes back to the trans-Golgi network (TGN) is important for maintaining protein homeostasis, recycling receptors, and returning molecules that were transported to the wrong compartments. Two important transmembrane proteins directed to this pathway are the Cation-Independent Mannose-6-phosphate receptor (CI-MPR) and the ATP7B copper transporter. Among CI-MPR functions is the delivery of acid hydrolases to lysosomes, while ATP7B facilitates the transport of cytosolic copper ions into organelles or the extracellular space. Precise subcellular localization of CI-MPR and ATP7B is essential for the proper functioning of these proteins. This study shows that both CI-MPR and ATP7B interact with a variant of the clathrin adaptor 1 (AP-1) complex that contains a specific isoform of the γ-adaptin subunit called γ2. Through synchronized anterograde trafficking and cell-surface uptake assays, we demonstrated that AP-1γ2 is dispensable for ATP7B and CI-MPR exit from the TGN while being critically required for ATP7B and CI-MPR retrieval from endosomes to the TGN. Moreover, AP-1γ2 depletion leads to the retention of endocytosed CI-MPR in endosomes enriched in retromer complex subunits. These data underscore the importance of AP-1γ2 as a key component in the sorting and trafficking machinery of CI-MPR and ATP7B, highlighting its essential role in the transport of proteins from endosomes.

Lysosomal dysfunction has been found in many pathological conditions, and methods to improve lysosomal function have been reported to be protective against infarcted hearts. However, the mechanisms underlying lysosomal dysfunction caused by ischemic injury are far less well-established. The retromer complex is implicated in the trafficking of cation-independent mannose 6-phosphate receptor (CI-MPR), which is an important protein tag for the proper transport of lysosomal contents and therefore is important for the maintenance of lysosomal function. In this study, we found that the function of retrograde transport in cardiomyocytes was impaired with ischemia/hypoxia (I/H) treatment, which resulted in a decrease in CI-MPR and an abnormal distribution of lysosomal cathepsins. I/H treatment caused a reduction in TBC1D5 and a blockade of the Rab7 membrane cycle, which impeded retromer binding to microtubules and motor proteins, resulting in an impairment of retrograde transport and a decrease in CI-MPR. We also established that TBC1D5 was an important regulator of the distribution of lysosomal cathepsins. Our findings shed light on the regulatory role of retromer in ischemic injury and uncover the regulatory mechanism of TBC1D5 over retromer.

The Golgi-localized, gamma-ear containing, ADP-ribosylation factor-binding proteins (GGAs 1, 2, and 3) are multidomain proteins that bind mannose 6-phosphate receptors (MPRs) at the Golgi and play a role, along with adaptor protein complex 1 (AP-1), in the sorting of newly synthesized lysosomal hydrolases to the endolysosomal system. However, the relative importance of the two types of coat proteins in this process is still unclear. Here, we report that inactivation of all three GGA genes in HeLa cells decreased the sorting efficiency of cathepsin D from 97% to 73% relative to wild-type, with marked redistribution of the cation-independent MPR from peripheral punctae to the trans-Golgi network. In comparison, GNPTAB-/- HeLa cells with complete inactivation of the mannose 6-phosphate pathway sorted only 20% of the cathepsin D. We conclude that the residual sorting of cathepsin D in the GGA triple-knockout cells is mediated by AP-1.

The cation-independent mannose 6-phosphate (M6P)/Insulin-like growth factor-2 receptor (CI-MPR/IGF2R) is an ∼300 kDa transmembrane protein responsible for trafficking M6P-tagged lysosomal hydrolases and internalizing IGF2. The extracellular region of the CI-MPR has 15 homologous domains, including M6P-binding domains (D) 3, 5, 9, and 15 and IGF2-binding domain 11. We have focused on solving the first structures of human D7-10 within two multi-domain constructs, D9-10 and D7-11, and provide the first high-resolution description of the high-affinity M6P-binding D9. Moreover, D9 stabilizes a well-defined hub formed by D7-11 whereby two penta-domains intertwine to form a dimeric helical-type coil via an N-glycan bridge on D9. Remarkably the D7-11 structure matches an IGF2-bound state of the receptor, suggesting this may be an intrinsically stable conformation at neutral pH. Interdomain clusters of histidine and proline residues may impart receptor rigidity and play a role in structural transitions at low pH.

Human retromer, a heterotrimer of VPS26 (VPS26A or VPS26B), VPS35 and VPS29, orchestrates the endosomal retrieval of internalised cargo and promotes their cell surface recycling, a prototypical cargo being the glucose transporter GLUT1 (also known as SLC2A1). The role of retromer in the retrograde sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR, also known as IGF2R) from endosomes back to the trans-Golgi network remains controversial. Here, by applying knocksideways technology, we develop a method for acute retromer inactivation. While retromer knocksideways in HeLa and H4 human neuroglioma cells resulted in time-resolved defects in cell surface sorting of GLUT1, we failed to observe a quantifiable defect in CI-MPR sorting. In contrast, knocksideways of the ESCPE-1 complex - a key regulator of retrograde CI-MPR sorting - revealed time-resolved defects in CI-MPR sorting. Together, these data are consistent with a comparatively limited role for retromer in ESCPE-1-mediated CI-MPR retrograde sorting, and establish a methodology for acute retromer and ESCPE-1 inactivation that will aid the time-resolved dissection of their functional roles in endosomal cargo sorting.

Cation-dependent mannose 6-phosphate receptor (CD-MPR) and cation-independent MPR (CI-MPR) belong to the P-type lectin family. Both intracellular and cell surface MPRs can recognize and bind with the terminal mannose 6-phospahte (M6P) residues of N-glycans attached to the mammalian lysosomal enzymes and the related co-factors. Domain9 (Dom9), which is one of the extracytoplasmic region of the CI-MPR, has relatively higher affinity for M6P residues. Here we describe the production of recombinant Dom9-His protein by Pichia pastris, purification, and application as a probe for lectin blotting.

DRAM1 (DNA damage-regulated autophagy modulator 1) is a transmembrane protein that predominantly localizes to the lysosome but is also found in other membranous organelles; however, its function in these organelles remains largely unknown. We found that DRAM1 was partially located in the Golgi apparatus, and knockdown of DRAM1 caused fragmentation of the Golgi apparatus in cells. The phenomenon of fragmented Golgi was not related to microtubule organization, and there was no direct interaction between DRAM1 and Golgi structural proteins (ARF1, GM130, syntaxin 6 and GRASP55). Moreover, Golgi-targeting DRAM1 failed to rescue the fragmentation of Golgi in DRAM1-deficient cells. The transport of ts045-VSVG-GFP, an indicator of movement from the Golgi apparatus to the plasma membrane, was delayed in DRAM1-knockdown cells. Moreover, the trafficking of CI-MPR from the plasma membrane to the Golgi was also impeded in DRAM1-knockdown cells. These results indicated that DRAM1 regulated the structure of the Golgi apparatus and affected Golgi apparatus-associated vesicular transport.

Palmitate attenuates insulin secretion and reduces the viability of insulin-producing cells. Previous studies identified the aberrant palmitoylation or mispalmitoylation of proteins as one mechanism by which palmitate causes β-cell damage. In this report, we identify a role for lysosomal protein degradation as a mechanism by which β cells defend themselves against excess palmitate. The cation-independent mannose 6-phosphate receptor (CI-MPR) is responsible for the trafficking of mannose 6-phosphate-tagged proteins to lysosomes via Golgi sorting and from extracellular locations through endocytosis. RINm5F cells, which are highly sensitive to palmitate, lack CI-MPR. The reconstitution of CI-MPR expression attenuates the induction of endoplasmic reticulum (ER) stress and the toxic effects of palmitate on RINm5F cell viability. INS832/13 cells express CI-MPR and are resistant to the palmitate-mediated loss of cell viability. The reduction of CI-MPR expression increases the sensitivity of INS832/13 cells to the toxic effects of palmitate treatment. The inhibition of lysosomal acid hydrolase activity by weak base treatment of islets under glucolipotoxic conditions causes islet degeneration that is prevented by the inhibition of protein palmitoylation. These findings indicate that defects in lysosomal function lead to the enhanced sensitivity of insulin-producing cells to palmitate and support a role for normal lysosomal function in the protection of β cells from excess palmitate.

Proper intracellular cholesterol trafficking is critical for cellular function. Two lysosome-resident proteins, NPC1 and NPC2, mediate the egress of low-density lipoprotein-derived cholesterol from lysosomes. However, other proteins involved in this process remain largely unknown. Through amphotericin B-based selection, we isolated two cholesterol transport-defective cell lines. Subsequent whole-transcriptome-sequencing analysis revealed two cell lines bearing the same mutation in the vacuolar protein sorting 53 (Vps53) gene. Depletion of VPS53 or other subunits of the Golgi-associated retrograde protein (GARP) complex impaired NPC2 sorting to lysosomes and caused cholesterol accumulation. GARP deficiency blocked the retrieval of the cation-independent mannose 6-phosphate receptor (CI-MPR) to the trans-Golgi network. Further, Vps54 mutant mice displayed reduced cellular NPC2 protein levels and increased cholesterol accumulation, underscoring the physiological role of the GARP complex in cholesterol transport. We conclude that the GARP complex contributes to intracellular cholesterol transport by targeting NPC2 to lysosomes in a CI-MPR-dependent manner.

The small GTPase Rab9 has long been described as a protein that mediates endosome-to-trans-Golgi Network (TGN) transport, and specifically mannose-6-phospate receptor (MPR) recycling. However, studies have challenged this view by showing that Rab9 also is connected to sorting pathways toward the endolysosomal compartments. We recently characterized the spatio-temporal dynamics of Rab9 and, by using live cell imaging, we showed that it enters the endosomal pathway together with CI-MPR at the transition stage between early, Rab5-positive, and late, Rab7a-positive, endosomes. More so, the Rab9 constitutively active mutant, Rab9Q66L, accumulates on late endosomes and promotes carrier formation at the TGN. Here, we discuss our findings in light of previous reports on Rab9 in the retrograde transport pathway.