Segregation in the Golgi complex precedes export of endolysosomal proteins in distinct transport carriers

Abstract

Biosynthetic sorting of newly synthesized transmembrane cargos to endosomes and lysosomes is thought to occur at the TGN through recognition of sorting signals in the cytosolic tails of the cargos by adaptor proteins, leading to cargo packaging into coated vesicles destined for the endolysosomal system. Here we present evidence for a different mechanism in which two sets of endolysosomal proteins undergo early segregation to distinct domains of the Golgi complex by virtue of the proteins' luminal and transmembrane domains. Proteins in one Golgi domain exit into predominantly vesicular carriers by interaction of sorting signals with adaptor proteins, but proteins in the other domain exit into predominantly tubular carriers shared with plasma membrane proteins, independently of signal-adaptor interactions. These findings demonstrate that sorting of endolysosomal proteins begins at an earlier stage and involves mechanisms that partly differ from those described by classical models.

Figure 1.

Structure, localization, and ER exit of RUSH reporter proteins. (a) Schematic representation of streptavidin–KDEL “hook” and TfR, LAMP1, and CD-MPR “reporter” proteins used in the RUSH experiments. FP, fluorescent protein (GFP or mCherry). In all figures, green and red lettering corresponds to constructs tagged with GFP and mCherry, respectively. (b) RUSH imaging series of three reporter cargos, TfR, LAMP1, and CD-MPR, expressed in HeLa cells, from Video 1. Before the addition of biotin (time 0), the three cargos exhibit a typical ER localization. At 21 min after biotin addition, the cargos localize to the Golgi. At later times, they exit the Golgi, reaching their final destination after 60 min. Bars, 5 µm. (c) Kinetics of trafficking of RUSH cargos through the Golgi complex. The normalized intensity of the masked perinuclear region indicated in b was measured across the whole time course and plotted as a function of time. Values are mean ± SEM; n = 12 cells for each cargo. Notice that the three reporter proteins released from the ER are transported into the Golgi complex at about the same time.

Figure 2.

Endolysosomal proteins are exported from the Golgi complex in two distinct populations of transport carriers. (a–c) HeLa cells coexpressing streptavidin–KDEL with each of the indicated reporter proteins were treated with biotin and imaged live by spinning-disk confocal microscopy. The left panel shows single frames captured at the indicated times after addition of biotin. The right panel shows magnified images of the boxed areas. (d–f) HeLa cells coexpressing streptavidin–KDEL with combinations of the indicated reporter constructs were analyzed as in a–c. The left columns show single frames captured at the indicated times after addition of biotin (from Video 2 in d and Video 3 in e). The right columns show magnified images of the boxed areas. Bars: (low magnification) 5 µm; (high magnification) 1 µm.

Figure 3.

Segregation of endolysosomal proteins in the Golgi complex. (a–f) HeLa cells coexpressing streptavidin–KDEL with combinations of the indicated reporter proteins were fixed 30 min after the addition of biotin and imaged by Airyscan microscopy. (a and d) Golgi complexes of representative cells. (b and e) Magnified views of box 1. (c and f) Magnified views of box 2 and plots of fluorescence intensity along the white dashed lines. (g and h) HeLa cells coexpressing streptavidin–KDEL with combinations of the indicated reporter proteins were imaged live by Airyscan microscopy. The top rows show Golgi complexes from representative cells. The middle rows show magnifications of the boxed region. The bottom row shows plots of fluorescence intensity along the white dashed lines. Bars, 1 µm. (i) Pearson’s coefficients (r) of data sets of which g and h are representative. Values were normalized to 1.0 at the first time point and are represented as mean ± SEM (n = 7 cells for each pair of cargos). ns, not significant; *, P < 0.1; **, P < 0.01; ***, P < 0.001. Times indicated in g–i are normalized to observable initiation of tubule budding, allowing comparative statistics.

Figure 4.

Role of adaptor-binding motifs in export and segregation of endolysosomal proteins at the Golgi complex. (a) Sequences from the cytosolic domains of TfR, LAMP1, and CD-MPR. Motifs that bind to AP complexes in each protein are highlighted in red. Mutations are indicated with blue letters. (b and c) HeLa cells coexpressing streptavidin–KDEL together with TfR and LAMP1 reporter constructs having mutations in AP-binding motifs, namely TfR-Y20A/GDNS^31-34AAAA (b) or LAMP1-Y404A (c), were imaged live by spinning-disk confocal microscopy. Images are single frames from Video 9. The times after addition of biotin are indicated. (Right) Magnifications of the boxed regions. Bars: (low magnification) 5 µm; (high magnification) 1 µm. (d and e) HeLa cells coexpressing streptavidin–KDEL together with GGA1–GFP and CD-MPR (d) or CD-MPR-L274A/L275A (e) reporter proteins were fixed 30 min after the addition of biotin and imaged by Airyscan microscopy. Bar, 2 µm. Arrows indicate carriers containing GGA1–GFP. (f) Airyscan microscopy of HeLa cells coexpressing streptavidin–KDEL together with CD-MPR-L274A/L275A and TfR-Y20A/GDNS^31-34AAAA reporter proteins 30 min after the addition of biotin. The inset shows magnified images of the boxed regions. Bars, 1 µm.

Figure 5.

AP complexes are dispensable for cargo sorting into Golgi-derived tubular carriers. (a) Schematic representation of AP-1, AP-2, AP-3, and AP-4. (b) Confirmation of KO by immunoblot analysis of endogenous targets. Notice that AP-2 μ2 KO is not complete. Cells with complete KO of AP-2 μ2 were not found in the screening. WB, Western blotting. (c) Images from spinning-disk, live-cell microscopy of LAMP1 or TfR reporter proteins in AP-KO cell lines at the indicated times after biotin addition. Tubular carriers containing LAMP1 or TfR reporters were found in all of the AP-KO cells. Bars: 10 µm; (insets) 1 μm. (d) The LAMP1 reporter protein was expressed in each AP-KO cell line. Cells were fixed 60 min after the addition of biotin and stained for an endogenous lysosomal marker (LAMTOR4) to assess the requirement of AP complexes for transport to lysosomes. Bars: 5 µm; (insets) 1 µm.

Figure 6.

The luminal and transmembrane domains of endolysosomal proteins determine their intra-Golgi segregation. (a) Schematic representation of chimeric proteins generated by swapping luminal, transmembrane, and cytosolic domains from LAMP1 (L) and CD-MPR (M). The chimeras were fused to a fluorescent protein and SBP for use as reporter proteins in the RUSH system (Fig. 1 a). (b) HeLa cells coexpressing streptavidin–KDEL together with the indicated chimeras and TfR as reporter proteins were fixed 30 min after the addition of biotin and analyzed by Airyscan microscopy. Bars, 1 µm. Images show the presence or absence of the chimeras in tubular carriers emanating from the Golgi complex. (c–h) Cells in b were similarly imaged for the distribution of the chimeras in the Golgi complex. r, Pearson’s coefficient. Magnified images and plots of fluorescence intensity along the whited dashed lines are shown at right. Bars, 1 µm.

Figure 7.

Model depicting the sorting of endolysosomal proteins in the Golgi complex. Endolysosomal proteins are delivered from the ER to the Golgi complex in the same transport carriers. Once in the Golgi complex, sets of endolysosomal proteins segregate to distinct domains. One domain gives rise to tubular carriers in which endolysosomal and plasma membrane proteins leave the Golgi independently of cytosolic sorting signals and AP complexes. The other domain is the source of vesicular carriers into which endolysosomal proteins are sorted through interaction of cytosolic sorting signals with GGA proteins.