Full-length hTMEM59L cDNA was amplified from the human brain cDNA library (BioDev) and was cloned into pEGFP-N2 between the EcoRI and SmaI sites using the primers 5’-GTCGAATTCACCATGGCTGCGGTGGCGCT-3’ and 5’-GCACCCGGGCAGCTTGGTCAGGTCCAGC-3’ to generate a plasmid that expressed hTMEM59L-enhanced green fluorescent protein (EGFP). The hTMEM59L fragment was amplified from a U251 cDNA library and sequenced. To generate the TMEM59L mutant with a C-terminal deletion (cTMEM59L) and then cloned in pEGFP-N2 expression vector to generate a plasmid expressing a cTMEM59L-EGFP fusion protein was preserved in the laboratory. Full-length mouse TMEM59L (mTMEM59L) was PCR-amplified from a mouse brain cDNA library and cloned in pEGFP-N2 between the EcoRI and BamHI restriction sites using primers 5’-GTCGAATTCACCATGGCTGCGGTAGCATTGC-3’ and 5’-GCAGGATCCGCAGCGTGGTCAAATCAAGC-3’. Human APP was PCR amplified using the primers 5’-GACAAGCTTATGCTGCCCGGTTTGG-3’ and 5’-GATCCCGGGGTTCTGCATCTGCTCAAAGAACT-3’ and cloned into pEGFP-N2 at the HindIII and SmaI restriction sites. Human GDI1 was PCR amplified using the primers 5’-GATAAGCTTATGGACGAGGAATACGAT-3’ and 5’-GTAGGATCCGCTGCTCAGCTTCTCCAAAG-3’ and cloned into pEGFP-N2 at the HindIII and BamHI sites. Human GDI2 was PCR amplified using the primers 5’-GATCTCGAGATGAATGAGGAGTACGAC-3’ and 5’-GTAGGATCCGGTCTTCCCCATAGATGTCA-3’ and cloned into pEGFP-N2 between the Xho I and BamHI sites. Human BACE2 was PCR amplified using the primers 5’-GATCTCGAGATGGGCGCACTGGC-3’ and 5’-GTAGGATCCGTTTCCAGCGATGTCTGACC-3’ and cloned into pEGFP-N2 at the Xho I and BamHI sites. In addition, hTMEM59L and mTMEM59L were also cloned into pcDNA3.1myc/his(A). hTMEM59L was additionally cloned into pDsRed-Express-N1. PCR products digested with two enzymes were purified using the Axyprep^TM PCR Cleanup kit (AxyGEN, USA). RNA was isolated using the RNAiso^TM Plus kit according to the manufacturer’s specifications. The RNA concentration was determined by measuring the absorbance ratio at 260/280 nm using a spectrophotometer (Eppendorf, Hamburg, Germany). cDNA was synthesized by reverse-transcribing 5 µg of total RNA using ReverTra Ace, and oligo (dT) were used for first-stand synthesis following the manufacturer’s instructions. Then DNase I was added to the system and incubated at 37°C for 30 min and stored at −80°C until used.

U251 and U87 human glioma cells, human embryonic kidney (HEK) 293T cells, 4T1 mouse mammary tumor cells, and U87 human glioma cells were purchased from the Chinese Academy of Sciences (Shanghai, China). U251, HEK293T, and U87 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) with 10% fetal calf serum (FCS, Gibco). 4T1 cells were cultured in Roswell Park Memorial Institute-1640 (Gibco) medium with 10% FCS. The C17.2 mouse neural stem cell (NSC) line was cultured in high-glucose DMEM containing 10% FCS and 5% horse serum (Gibco). All cells were cultured in a humidified atmosphere containing 5% CO₂ at 37°C. U251, HEK293T, 4T1, U87, and C17.2 cells were transiently transfected with respective plasmid constructs using Lipofectamine 2000 reagent (Invitrogen) when they reached 60%–70% confluency. Cells were exposed to transfection complexes for 4 h in a serum-free medium, and then a normal culture medium was added, in accordance with the manufacturer’s instructions.

Twenty-four hours after transfection of the vectors containing EGFP-tagged TMEM59L, cells were washed three times with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 30 min, and then washed again with PBS. U251 cells were also transfected with vTMEM59L and cTMEM59L. Nuclei were stained with DAPI for 5 min at room temperature. Golgi-Tracker Red, Lyso-Tracker Red, and Mito-Tracker Green (Beyotime Biotechnology Company, China) were used for staining. U251 and C17.2 cells were transiently transfected with hTMEM59L-EGFP for 24 h and then stained with Golgi-Tracker Red or Lyso-Tracker Red to label the Golgi apparatus and acidic organelles, respectively. U251 cells were transiently transfected with hTMEM59L-DsRed for 24 h and then stained with Mito-Tracker Green to stain the mitochondria.

To analyze the dynamics of TMEM59L in live cells, fluorescence was observed at 48 h after transfection of plasmids using a Nikon Eclipse TE2000-U fluorescence microscope. Images were acquired using a SPOT CCD digital camera.

HEK293T cells were co-transfected with EGFP-APP, EGFP-GDI1, EGFP-GDI2, EGFP-BACE2, together with pcDNA3.1myc/his(A), pcDNA3.1myc/his(A)-hTMEM59L, or pcDNA3.1myc/his(A)-mTMEM59L. Cells were harvested by scraping, washed with cold PBST (PBS containing 0.5% Tween 20), and incubated in lysis buffer according to the manufacturer’s instructions (KeyGEN BioTECH, Nanjing, China). Samples were mixed with protein loading buffer, boiled for 5 min, electrophoresed in 12% sodium dodecyl sulfate-polyacrylamide gels, and transblotted to cellulose nitrate membranes (Whatman, Maidstone, Kent, UK) for 1 h at 25 V. Membranes were blocked with PBST containing 1% bovine serum albumin at room temperature and then incubated with an anti-GFP, anti-myc, or anti-actin antibody (1:2000; Santa Cruz) for 2 h at room temperature. Thereafter, membranes were rinsed, incubated with a secondary antibody conjugated with a far-infrared dye (1:12000; LI-COR, Lincoln, NE, USA) for 1 h, and scanned using an Odyssey scanner (LI-COR).

U251, HEK293T, C17.2, 4T1, and U87 cells were transfected with a plasmid harboring hTMEM59L-EGFP. The pEGFP-N2 plasmid was used as a negative control. hTMEM59L localized to vesicular structures in the cytoplasm and perinuclear region but was not detected in the nucleus (Fig. 1).

Twenty colony clones of full-length hTMEM59L were rescreened by PCR, one of which was a splice variant comprising exons 1, 2, 3, and 8 of hTMEM59L (named vTMEM59L). vTMEM59L contains 534 nucleotides and encodes a protein of 178 amino acids, meaning that a middle region of the hTMEM59L gene was deleted (Supplement Fig. 1. To determine which regions of hTMEM59L are functionally important, we deleted the C-terminal region to generate cTMEM59L. This mutant is 808 nucleotides long and encodes a protein of 269 amino acids (Supplement Fig. 2). Fluorescence was detected in U251 cells transfected with vTMEM59L, and cTMEM59L, indicating the expression of proteins; however, the fluorescence intensity was markedly lower than in cells transfected with hTMEM59L. More importantly, the number of vesicular structures was markedly lower in cells transfected with vTMEM59L and cTMEM59L than in cells transfected with hTMEM59L, illustrating that the middle and C-terminal regions of hTMEM59L are crucial for its expression and localization (Fig. 2).

The subcellular localization of hTMEM59L in U251 and C17.2 cells was further assessed by staining cells with Golgi-Tracker Red and Lyso-Tracker Red dyes and observed that hTMEM59L mainly localized in lysosomes and acidic vesicular structures. Dynamic punctuated fluorescent signals were observed in the neurites and cytoplasm of living U251 cells transiently transfected with the plasmid harboring hTMEM59L-EGFP (Fig. 3).

In control transfected HEK293T cells, EGFP-APP fluorescence was observed in the cytoplasm and the Golgi bodies. In contrast, in cells transfected with TMEM59L, EGFP-APP fluorescence in the Golgi bodies significantly decreased. Further co-localization analysis showed significant localization of APP and TMEM59L to Golgi bodies (Fig. 4). HEK293T cells were transiently co-transfected with GDI1 and TMEM59L, and a significantly enhanced fluorescence intensity was observed and the cells appeared more spherical. Co-localization studies showed that GDI1 distribution was not significantly affected by TMEM59L (Fig. 5). However, western blotting demonstrated that overexpression of TMEM59L markedly increased the level of APP protein. Overexpression of TMEM59L protein further increased GDI1 and GDI2 levels but did not affect BACE2 levels (Figs. 6, 7, Supplement Fig. 3).