Figure 1.
Stability of NPC2-mCherry fusion proteins.
A) Design of fusion proteins and schematic of mCherry. Fluorescent tags were engineered to the C-termini of proteins of interest. The N-terminal 11 amino acid region of mCherry (MVSKGEEDNMA) was deleted to create crmCherry. In the mCherry structure shown, residues 1–11 are shown in red. B) Npc2−/− MEFs, U2OS, and CHO cells were transiently transfected with constructs expressing mCherry alone, NPC2 alone or an NPC2-mCherry fusion with a 9aa linker sequence (RARDPPVAT). After 48 h, cell lysates were analyzed by immunoblotting for NPC2 or mCherry. β-actin was used as a loading control. C) Schematic illustrating the indicated numbered bands shown in Panel B.
Figure 2.
Effect of linker region on stability of NPC2 fusion proteins.
Npc2−/− MEFs, U2OS, and CHO cells were transiently transfected with a vector control or indicated fusion constructs. Linker sequences are as described in Fig. 1 legend, and below: 5aa: (PPVAT), P5: (PPPPP), P4: (PPPP), P3: (PPP), P2: (PP), P1: (P). After 48 h, cell lysates were analyzed by immunoblotting as in Fig. 1.
Figure 3.
Subcellular localization of NPC2-crmCherry fusion proteins.
A) U2OS cells were transiently transfected with indicated fusion constructs. Linker sequences are as described in Fig. 2 legend and below: P10: (PPPPPPPPPP); (GS)5, (GSGSGSGSGS). Endocytosed Alexa488-NPC2 is used as a lysosomal standard. The scale bar (white) in the bottom right corner represents 20 µm. (B) Quantitative co-localization analysis. Mander’s colocalization coefficients (tM) were plotted for individual fields of cells. Black dots (tM1) represent Mander’s coefficients for the fraction of pixels containing red signal from crmCherry/mCherry that also contained signal from green-NPC2, while red dots (tM2) represent Mander’s coefficients for the fraction of lysosomes (marked by green-NPC2) that also contain red fluorescent protein chimera. Each point represents analysis of a single field containing 1–4 transfected cells. The number of cells analyzed for each fusion construct are as follows: NPC2-crM (22), NPC2-P5-crM (45), NPC2-P10-crM (29), NPC2-GSGSG-crM (28), NPC2-(GS)5-crM (39), NPC2-mCherry (34).
Figure 4.
Function of the NPC2-crmCherry fusion protein.
Npc2−/− MEF cells were stably transfected with the NPC2-crmCherry fusion construct and compared to non-transfected control. Cholesterol accumulation was visualized by staining with filipin.
Figure 5.
Linker sequences affect lysosomal targeting of TPP1 fusion proteins.
A) U2OS cells were transiently transfected with indicated fusion constructs. Linker sequences are as described in Fig. 3 legend. Endocytosed Alexa488-NPC2 is used as a lysosomal standard. The scale bar (white) in the bottom right corner represents 20 µm. B) Quantitative co-localization analysis. Mander’s colocalization coefficients (tM) were plotted for individual fields of cells. Each dot represents analysis of a single field containing 1–4 transfected cells. Black dots (tM1) represent Mander’s coefficients for the fraction of pixels containing red signal from crmCherry/mCherry that also contained signal from green-NPC2, while red dots (tM2) represent Mander’s coefficients for the fraction of lysosomes (marked by green-NPC2) that also contain red fluorescent protein chimera. The number of cells analyzed for each fusion construct are as follows: TPP1-crM (33), TPP1-P5-crM (37), TPP1-P10-crM (43), TPP1-GSGSG-crM (43), TPP1-(GS)5-crM (34), TPP1-mCherry (33).
Figure 6.
Expression of TPP1 fusion proteins.
CHO cells were transiently transfected as indicated. Media was collected at 24) TPP1 and β-galactosidase activity measured in media and cell lysates. Data represents total activity in media (24 and 48-hour collection points) and cells. B) Blot analysis of cell lysates and 48-hour collection point for media samples. Note that ∼5 times greater proportional equivalence of cell lysate was loaded compared to media samples. C) Illustration of processing of TPP1 fusion proteins in the lysosome observed in Panel B. Glycans are labeled with or without P to indicate removal of the M6P modification. Note that this is for heuristic purposes only and does note imply stoichiometry or sites of phosphorylation.
Figure 7.
M6P content of TPP1 fusion proteins.
CHO cells were transiently transfected with the indicated constructs. The media samples collected at the two time points as in Fig. 6 were pooled and used for MPR-affinity chromatography. Fractions (1, flow through; 2 and 3, washes; 4 and 5, G6P/mannose mock elution; 6–8, specific elution with M6P; 9 and 10, glycine elution) were analyzed for TPP1 activity after preactivation.
Figure 8.
Modeling of fusion protein constructs.
Models of NPC2 (A) and TPP1 (B) directly fused to crmCherry, or separated by Pro or Ser and Gly linkers, were generated by merging protein structures with the low energy configurations of high mannose oligosaccharides placed at the common coordinates of GlcNAc groups. For TPP1, the oligosaccharides linked to Asn286 and Asn313 are closest to mCherry and are oriented pointing up and down, respectively. Envelopes of mCherry represent 50 randomly chosen structures from the allowable modeled conformations.
Table 1.
Allowable number of linker conformations in the absence and presence of fusion proteins*.
Figure 9.
Intracellular localization of ARSK fusion proteins.
A) U2OS cells were transiently transfected with indicated fusion constructs. Linker sequences are as described in Fig. 3 legend. Endocytosed Alexa488-NPC2 is used as a lysosomal standard. The scale bar (white) in the bottom right corner represents 20 µm. B) Quantitative co-localization analysis. Mander’s colocalization coefficients (tM) were plotted for individual fields of cells. Each dot represents analysis of a single field containing 1–4 transfected cells. Black dots (tM1) represent Mander’s coefficients for the fraction of pixels containing red signal from crmCherry/mCherry that also contained signal from green-NPC2, while red dots (tM2) represent Mander’s coefficients for the fraction of lysosomes (marked by green-NPC2) that also contain red fluorescent protein chimera. The number of cells analyzed for each fusion construct are as follows: ARSK-crM (10), ARSK-P5-crM (16), ARSK-P10-crM (12), ARSK-GSGSG-crM (20), ARSK-(GS)5-crM (13), ARSK-mCherry (11).
Figure 10.
Intracellular localization of FUCA2 fusion proteins.
A) U2OS cells were transiently transfected with indicated fusion constructs. Linker sequences are as described in Fig. 3 legend. Endocytosed Alexa488-NPC2 is used as a lysosomal standard. The scale bar (white) in the bottom right corner represents 20 µm. B) Quantitative co-localization analysis. Mander’s colocalization coefficients (tM) were plotted for individual fields of cells. Each dot represents analysis of a single field containing 1–4 transfected cells. Black dots (tM1) represent Mander’s coefficients for the fraction of pixels containing red signal from crmCherry/mCherry that also contained signal from green-NPC2, while red dots (tM2) represent Mander’s coefficients for the fraction of lysosomes (marked by green-NPC2) that also contain red fluorescent protein chimera. The number of cells analyzed for each fusion construct are as follows: FUCA2-crM (13), FUCA2-P5-crM (15), FUCA2-P10-crM (12), FUCA2-GSGSG-crM (13), FUCA2-(GS)5-crM (20), FUCA2-mCherry (16).
Figure 11.
Intracellular localization of LPO fusion proteins.
A) U2OS cells were transiently transfected with indicated fusion constructs. Linker sequences are as described in Fig. 3 legend. Endocytosed Alexa488-NPC2 is used as a lysosomal standard. The scale bar (white) in the bottom right corner represents 20 µm. B) Quantitative co-localization analysis. Mander’s colocalization coefficients (tM) were plotted for individual fields of cells. Each dot represents analysis of a single field containing 1–4 transfected cells. Black dots (tM1) represent Mander’s coefficients for the fraction of pixels containing red signal from crmCherry/mCherry that also contained signal from green-NPC2, while red dots (tM2) represent Mander’s coefficients for the fraction of lysosomes (marked by green-NPC2) that also contain red fluorescent protein chimera. The number of cells analyzed for each fusion construct are as follows: LPO-crM (30), LPO-P5-crM (22), LPO-P10-crM (12), LPO-GSGSG-crM (25), LPO-(GS)5-crM (22), LPO-mCherry (16).
Figure 12.
Intracellular localization of OS9 fusion proteins.
A) U2OS cells were transiently transfected with indicated fusion constructs. Linker sequences are as described in Fig. 3 legend. Endocytosed Alexa488-NPC2 is used as a lysosomal standard. The scale bar (white) in the bottom right corner represents 20 µm. B) Quantitative co-localization analysis. Mander’s colocalization coefficients (tM) were plotted for individual fields of cells. Each dot represents analysis of a single field containing 1–4 transfected cells. Black dots (tM1) represent Mander’s coefficients for the fraction of pixels containing red signal from crmCherry/mCherry that also contained signal from green-NPC2, while red dots (tM2) represent Mander’s coefficients for the fraction of lysosomes (marked by green-NPC2) that also contain red fluorescent protein chimera. The number of cells analyzed for each fusion construct are as follows: OS9-crM (10), OS9-P10-crM (12), OS9-(GS)5-crM (12).