Figure 4. Golgi enzymes primarily localize to the interior of medial and trans-Golgi cisternae.

(A, C and E) En face view images of Golgi enzymes. Side view images are also shown in (A) and (C). Dotted arrows and boxes and line intensity profiles are used or acquired as in Figure 1K. (B, D and F) Corresponding en face averaged images and radial mean intensity profiles. n, the number of averaged Golgi mini-stack images. (G) The merge of en face averaged images of Giantin, MGAT4B and MGAT2 and the corresponding radial mean intensity profile. n, the number of averaged Golgi mini-stack images. (H) β4GalT3 and ST6Gal1 can localize to shared (arrows) and distinct domains within the cisternal interior. (I) MGAT2 localizes to the cisternal interior of the native Golgi by EM. NRK cells transiently expressing MGAT2-APEX2-GFP were subjected to APEX2-catalyzed reaction followed by EM. Note that cells were not subjected to nocodazole treatment. The thin section EM image displays the side view of a Golgi stack. MGAT2-APEX2 positive cisternal interior and budding profiles are indicated by arrows and arrow heads, respectively. (J) A quantitative molecular map of the Golgi mini-stack. The normalized radius of a Golgi protein is plotted against its corresponding LQ (Table 1). Red open and closed circle denote ring and disk lateral localization pattern, respectively. n, the number of Golgi mini-stacks used to calculate normalized radius. (K,L) Identifying the rim and interior of native Golgi cisternae. Cells were not treated with nocodazole. In (K), the cisternal rim (arrows) and interior are labeled by Giantin and β4GalT3, respectively. In (L), Giantin and GPP130 positive curvy lines (arrows) represent cisternal rim and do not correspond to side views or cross sections of Golgi stacks. The boxed region in each image is enlarged in the upper right corner. Scale bars represent 500 nm unless specified otherwise.

Figure 4—figure supplement 1. Golgi enzymes that primarily display central disk localization at the interior of medial and trans-Golgi cisternae.

(A, C, E, G, I, K, M, O, Q and S) En face view images of Golgi enzymes and SLC35C1. Side view images are also shown in (A). Dotted arrows and boxes and line intensity profiles are used or acquired as in Figure 1K. (B, D, F, H, J, L, N, P, R and T) Corresponding en face averaged images and radial mean intensity profiles. n, the number of averaged mini-stack images. (U) MGAT2-APEX2 biotinylated proteins mainly localize to the cisternal interior. Cells expressing MGAT2-APEX2-GFP were subjected to APEX2-catalyzed reaction to biotinylate its neighboring proteins. The biotinylated proteins were detected by Alexa Fluor 594 conjugated streptavidin (streptavidin-594). The line intensity profile is generated as in Figure 1K. Scale bar, 500 nm.

Figure 4—figure supplement 2. Golgi enzymes, Man1B1, ManII and TPST2, display ring-pattern localization.

(A, C and E) En face view images. (B, D and F) Corresponding en face averaged images and radial mean intensity profiles. n, the number of averaged mini-stack images. The line intensity profile is generated as in Figure 1K. Scale bar, 500 nm.

Figure 4—figure supplement 3. MGAT2 mainly localizes to the cisternal interior in native Golgi stacks by EM.

(A) Transmitted light image of NRK cells expressing MGAT2-APEX2-GFP after APEX2-catalyzed labeling reaction and plastic embedding. (B) Representative overview EM image of a cell expressing MGAT2-APEX2-GFP. Scale bar, 2 µm. (C) Categorization of MGAT2-APEX2-GFP distribution patterns. The experiment is the same as in Figure 4I. 58 MGAT2-APEX2 positive Golgi stacks from 14 cells were imaged. Side and en face views of Golgi stacks are further categorized as interior and rim-localization according to their APEX2 staining patterns. Scale bar, 200 nm.