Golgi Distribution of Lyn to Caveolin- and Giantin-Positive cis-Golgi Membranes and the Caveolin-Negative, TGN46-Positive trans-Golgi Network

Abstract

Src-family tyrosine kinases, classified as cytosolic enzymes, have crucial roles in regulating cell proliferation, differentiation, migration and cell-shape changes. Newly synthesized Lyn, a member of Src-family kinases, is biosynthetically accumulated at the cytoplasmic face of caveolin-containing Golgi membranes via posttranslational lipid modifications and then transported to the plasma membrane. However, the precise intra-Golgi localization of Lyn remains elusive. By means of a 19°C block-release technique and short-term brefeldin A treatment, we show here that the distribution of Lyn is not monotonously spread within the Golgi but selectively intensified in two distinct membrane compartments: giantin- and caveolin-positive membranes and trans-Golgi network protein (TGN)46-positive but caveolin-negative membranes. Furthermore, Lyn exits the Golgi from the caveolin-positive cis-Golgi cisternae or the caveolin-negative trans-Golgi network. These results suggest that Lyn moves apart from caveolin, a secretory protein, within the Golgi during Lyn’s trafficking to the plasma membrane.

Src-family non-receptor-type tyrosine kinases include at least eight highly homologous proteins: Src, Lyn, Yes, Fyn, Fgr, Hck, Lck and Blk.^1,2) Src-family kinases play crucial roles in regulating cell proliferation, differentiation, migration and cell-shape changes.^1,2)

Src-family kinases, classified as cytosolic enzymes, are anchored to the cytoplasmic face of the plasma membrane through myristoylation and palmitoylation.³⁾ We showed that newly synthesized Lyn, a member of Src-family kinases, in the cytoplasm is biosynthetically accumulated at the Golgi, which contains various membranous cisternae and clusters of vesicles, including cis-, medial- and trans-Golgi cisternae and the trans-Golgi network (TGN),^4,5) and Golgi-associated Lyn is subsequently transported to the plasma membrane.^6–10) Since the localization of proteins is linked to their functions, we also showed that Golgi-associated Lyn can serve as a signaling platform under oxidative stress.⁷⁾ Although newly synthesized secretory proteins move through the secretory pathway: rough endoplasmic reticulum (ER)→cis-, medial- and trans-Golgi cisternae→secretory or transport vesicles→plasma membrane, the intra-Golgi localization of Lyn remains elusive.

In this study, we investigated the precise distribution of Lyn within the Golgi by means of temperature block and release together with brefeldin A or tannic acid treatment.

MATERIALS AND METHODS

Plasmids, Cells and Transfection

cDNA encoding human Lyn¹¹⁾ (provided by T. Yamamoto) was subcloned into the pcDNA4/TO vector (Invitrogen, U.S.A.).⁶⁾ cDNA encoding green fluorescent protein (GFP)-tagged neurotrophin receptor p75 (p75-GFP) was provided by E. Rodriguez-Boulan.¹²⁾ COS-1 cells were cultured in Iscove’s modified Dulbecco’s medium containing 5% bovine serum. Cells were transiently transfected with polyethelenimine and cultured for 6–8 h.¹³⁾

Antibodies

The following antibodies were used: Lyn (H-6; Santa Cruz Biotechnology, U.S.A. and Y497; Abcam, U.K.), caveolin (BD Biosciences, U.S.A.), giantin (G1/133, ALEXIS, U.S.A.), TGN46 (Serotec, U.S.A.), GM130 (#35, BD Biosciences), and CI-MPR (Thermo Scientific Pierce, U.S.A.), and β-1,4-galactosyltransferase¹⁴⁾ (provided by M. N. Fukuda). Tetramethylrhodamine-isothiocyanate (TRITC)-, Alexa Fluor 488-, Alexa Fluor 546- or Alexa Fluor 647-labelled secondary antibodies were purchased from Sigma and Invitrogen.

Immunofluorescence

Cells were fixed and stained, as described.^10,15) Confocal images were obtained using a Fluoview FV500 (Olympus, Japan) laser scanning microscope with a 60× water-immersion objective. The Pearson’s R values were determined as described.¹⁵⁾ At present, we do not have appropriate means to examine the intra-Golgi localization of endogenous Lyn because of the following reasons. In COS-1 cells, endogenous Lyn could not be visualized due to the low expression. In HeLa cells, endogenous Lyn was minimally visible in the Golgi despite the low expression,⁶⁾ but we were unable to visualize the precise localization of Lyn within the intra-Golgi. In THP-1 monocytic cells, endogenous Lyn was sufficiently visible in the Golgi,⁸⁾ but THP-1 cells were not appropriate for close examination of Lyn’s intra-Golgi localization because each THP-1 cell has a large nucleus and the Golgi shape is very thin in the narrow cytoplasm.

Temperature Block and Release

Cells transfected with Lyn were cultured at 37°C for 6 h and then shifted to 19°C for 2 h to accumulate proteins in the Golgi.^8,9) For release from a temperature block, cells were warmed to 37°C for 0, 15, 30, and 60 min in the presence of 200 µg/mL cycloheximide to inhibit protein synthesis. The addition of cycloheximide effectively creates a pulse of newly synthesized protein that can be chased synchronously from the Golgi to the plasma membrane.¹⁶⁾

Tannic Acid Treatment

After a temperature block at 19°C for 2 h, cells were treated with 0.5% tannic acid at 32°C for 1 h.^9,17)

RESULTS AND DISCUSSION

To characterize the localization of Lyn within the Golgi, we visualized several endogenous Golgi proteins and evaluated the colocalization levels by determining the Pearson’s R values. First, we compared Lyn with two cis-Golgi proteins: giantin and cis-Golgi matrix protein (GM) 130. The localization of Lyn in the Golgi was appreciably similar to that of giantin, whereas the localization of Lyn was distinct from that of GM130 (Fig. 1A), suggesting that Lyn is distributed to the Golgi membranes harboring giantin rather than GM130. Next, we performed a temperature block at 19°C to prevent vesicular trafficking through the trans-Golgi network and compared the localization of Lyn with that of two trans-Golgi proteins: trans-Golgi network protein (TGN)46 and cation-independent mannose-6-phosphate receptor (CI-MPR). Note that TGN46 mediates protein trafficking between the Golgi and the plasma membrane, whereas CI-MPR mediates the transport of lysosomal enzymes.¹⁸⁾ The localization and the R values showed that a large fraction of Lyn was colocalized with TGN46 but not with CI-MPR (Fig. 1B). Because TGN38/46 is closely linked to the golgin GCC88, which is required for efficient retrograde transport,¹⁹⁾ TGN46-colocalized Lyn may be involved in transport of TGN46.

Fig. 1. Lyn Localizes to Two Differential Areas in the Golgi

(A) Cells transfected with Lyn were cultured at 37°C for 8 h. Lyn and endogenous Golgi proteins were visualized with anti-Lyn, anti-giantin and anti-GM130 antibodies. (B) Cells transfected with Lyn were cultured at 37°C for 8 h and then shifted to 19°C for 2 h. Cells were doubly stained with anti-Lyn and anti-TGN46 antibodies or with anti-Lyn and anti-CI-MPR antibodies. (C) Cells transfected with Lyn were cultured at 37°C for 8 h and doubly stained with anti-Lyn and anti-caveolin antibodies. (D) Cells were cultured at 37°C for 6 h and then shifted to 19°C for 2 h. Cells were doubly stained with anti-caveolin and anti-giantin antibodies (upper) and anti-caveolin and anti-TGN46 antibodies (lower). Insets show magnified images of the squared areas. The Pearson’s R value was determined in the squared areas. Scale bars, 20 µm. (E) The plots represent the Pearson’s R values determined from individual cells in (A)–(D). The data represent the mean (in parentheses) ±S.D. (bar). The significant difference (***, p<0.001) is calculated by Student’s t test. n, cell number.

It is known that caveolin is synthesized in the ER, transported to the Golgi and then to the plasma membrane along the secretory pathway.²⁰⁾ Previously, we showed that Lyn is colocalized with the Golgi pool of caveolin.⁶⁾ Indeed, Lyn was nicely colocalized with caveolin in the Golgi (Fig. 1C). We further characterized Golgi membranes harboring Lyn and revealed that caveolin was not colocalized with TGN46 even at 19°C despite colocalization of caveolin with giantin (Fig. 1D), indicating that giantin-positive membranes are different from TGN46-positive membranes. Although we were unable to perform triple staining of Lyn, caveolin and giantin with the antibodies currently available because of the cross-reactivity of secondary antibodies, we showed the statistical data that were obtained from doubly stained cells to ensure Lyn’s intra-Golgi localization (Fig. 1E). These results suggest that Lyn is distributed to two Golgi membranes: caveolin- and giantin-positive membranes (triple colocalization of Lyn, caveolin, and giantin in the cis-Golgi) and TGN46-positive but caveolin-negative membranes (colocalization of Lyn and TGN46 in the trans-Golgi network).

We also showed that treatment with brefeldin A (BFA) for 1 h fully translocates Lyn from the Golgi to the ER,⁶⁾ because BFA causes the disassembly of the Golgi and the redistribution of Golgi proteins to the ER. However, the trans-Golgi network is relatively insensitive to BFA compared with the Golgi cisternae.^21,22) Given that Lyn was colocalized with TGN46 (Fig. 1B), we examined whether Lyn was distributed to the trans-Golgi network. After 5–10 min of BFA treatment, a fraction of Lyn still remained in the Golgi, but the trans-Golgi protein β-1,4-galactosyltransferase (GalT) disappeared from the Golgi (Fig. 2A). Furthermore, Lyn and TGN46 were found to be resistant to 5-min BFA treatment and they were partially colocalized (Fig. 2B). Despite colocalization of Lyn with caveolin and giantin (Figs. 1A, C), caveolin and giantin were dispersed by 5-min BFA treatment (Fig. 2C), suggesting that a fraction of Lyn in the Golgi is distributed to the trans-Golgi network other than the Golgi cisternae.

Fig. 2. Effect of BrefeldinA Treatment on Golgi Localization of Lyn

(A) Cells transfected with Lyn were cultured at 37°C for 8 h and subsequently incubated for 0–10 min in the presence of 3.5 µg/mL BFA. Cells were doubly stained with anti-Lyn and anti-GalT antibodies. (B, C) Cells transfected with Lyn were cultured at 37°C for 8 h and treated with 3.5 µg/mL BFA for 5 min. Cells were doubly stained with (B) anti-Lyn and anti-TGN46 antibodies and (C) anti-Lyn and anti-caveolin antibodies (top) or anti-Lyn and anti-giantin antibodies (bottom). Each panel shows a magnified image of the squared area. Scale bars, 20 µm.

The trans-Golgi network consists of tubular and vesicular membranes containing secretory proteins destined to be transported to the plasma membrane.^23,24) To visualize Lyn at the exit site of the Golgi, cells were cultured at 19°C for 2 h and released from the temperature block into 37°C medium in the presence of cycloheximide (CHX) to inhibit newly synthesis of Lyn (Fig. 3A). Following release from the temperature block, the level of Lyn at the Golgi was gradually decreased, suggesting that Lyn is departing from the Golgi toward the plasma membrane (Fig. 3B). Moreover, a remaining fraction of Lyn was still visible in a small area 60 min after release from the temperature block, and the fraction of Lyn became apart from giantin and TGN46. These results suggest that Lyn is apart from giantin- or TGN46-positive membranes when exiting the Golgi. Considering that recycling endosomes locate in the center of the Golgi ring in COS-1 cells²⁵⁾ and that Rab11 is localized to recycling endosomes²⁶⁾ and the dominant-negative mutant of Rab11 blocks the transport of Lyn to the plasma membrane⁸⁾ and endomembranes,¹⁵⁾ Lyn may be transported from giantin- or TGN46-positive membranes possibly to recycling endosomes that move to the plasma membrane.

Fig. 3. Time-Dependent Movement of Lyn within the Golgi

(A) Schematic depiction of temperature shifts to observe the movement of Lyn within the Golgi. (B) Cells transfected with Lyn were cultured at 37°C for 6 h and shifted to 19°C for 2 h to accumulate proteins in the Golgi. Subsequently, cells were incubated at 37°C for 0, 15, 30 or 60 min in the presence of 200 µg/mL cycloheximide (CHX) to inhibit protein synthesis, and then triply stained with anti-Lyn, anti-giantin, and anti-TGN46 antibodies. Each panel shows a magnified image of the Golgi. The Pearson’s R values were determined in the squared areas. Scale bars, 5 µm. (C) Cells transfected with Lyn and p75-GFP were cultured at 37°C for 7 h, shifted to 19°C for 2 h to accumulate Lyn and caveolin in the Golgi, and then treated with 200 µg/mL CHX to inhibit protein synthesis. Subsequently, cells were incubated at 32°C for 1 h in the presence of 0.5% tannic acid, which inhibits plasma membrane fusion. Cells were doubly stained with anti-Lyn and anti-caveolin antibodies. Insets containing PGCs show magnified images of the squared areas. Scale bars, 20 µm.

To compare the trafficking route for Lyn with those for caveolin and the neurotrophin receptor p75, which are secretory proteins and colocalized with giantin (Fig. 1D) and TGN46¹²⁾ respectively, post-Golgi transport carriers (PGCs) were visualized by tannic acid treatment. We found that Lyn-localized PGCs were different from p75-positive PGCs and caveolin-positive PGCs despite similar localization of Lyn, p75 and caveolin in the Golgi (Fig. 3C). These results suggest that Lyn is transported to the plasma membrane via a trafficking route that is distinct from those of p75 and caveolin.

In the present study, we demonstrate for the first time that the distribution of Lyn is not monotonously spread within the Golgi but selectively intensified in two distinct membrane compartments: giantin- and caveolin-positive cis-Golgi membranes and TGN46-positive but caveolin-negative trans-Golgi network membranes. As machinery for membrane trafficking through the Golgi is still controversial,²⁷⁾ we assume that Lyn, a cytosolic protein, exits the Golgi via a novel trafficking route other than the typical secretory pathway.

Acknowledgment

This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (21390017).

Conflict of Interest

The authors declare no conflict of interest.

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