Protein sorting in the secretory pathway is essential for maintaining cell compartmentalization and homeostasis. In addition to shell-mediated sorting, the role of lipids in kinesin sorting in the process of secretory transport is a long-standing basic question that has not yet been answered. Here, we perform 3D simultaneous multicolor high-resolution real-time imaging to prove in vivo that newly synthesized glycosylphosphatidylinositol-immobilized proteins with very long ceramide lipid moieties are clustered and classified into specialized endoplasms Net exit site, which is different from that used by transmembrane proteins. In addition, we show that the chain length of ceramide in the endoplasmic reticulum membrane is critical for this sorting selectivity. Our study provides the first direct in vivo evidence to classify protein cargoes based on lipid chain length into selective export sites in the secretory pathway.
In eukaryotic cells, the proteins synthesized in the endoplasmic reticulum (ER) are then sorted during transport through the secretory pathway for delivery to their appropriate cellular destination (1). In addition to coat-mediated sorting, it was long speculated that certain lipids can also serve as selective exit points by clustering them into specific membrane domains that specific proteins (2-5). However, there is still a lack of direct in vivo evidence to prove this possible lipid-based mechanism. In order to solve this basic problem, we studied in yeast how glycosylphosphatidylinositol (GPI) anchored proteins (GPI-APs) are differentially exported from the ER. GPI-APs are a variety of lipid-connected cell surface proteins ( 6, 7). GPI-AP is a secreted protein attached to the outer leaflets of the plasma membrane through the glycolipid moiety (GPI anchor). They accept GPI anchors as conservative post-translational modifications in the ER lumen (8). After attachment, GPI-AP passes through the Golgi apparatus (5, 9) from the ER to the plasma membrane. The presence of GPI anchors causes GPI-AP to be transported separately from transmembrane secreted proteins (including other plasma membrane proteins) along the secretory pathway (5, 9, 10). In yeast cells, GPI-APs are separated from other secreted proteins in the endoplasmic reticulum, and then packaged into unique vesicles wrapped by coat protein complex II (COPII) (6, 7). The determinants of this classification process in the ER export process are unclear, but it is speculated that this mechanism may require lipids, especially the structural remodeling of the lipid portion of the GPI anchor (5, 8). In yeast, GPI lipid remodeling begins immediately after the GPI attaches, and in many cases, it causes ceramide to bind to the 26-carbon long-chain saturated fatty acid (C26:0) (11, 12). C26 ceramide is the main ceramide produced by yeast cells so far. It is synthesized in the ER and most of it is exported to the Golgi apparatus through COPII vesicles (13). The ER export of GPI-AP specifically requires ongoing ceramide synthesis (14, 15), and in turn, the conversion of ceramide to inositol phosphate ceramide (IPC) in the Golgi apparatus depends on GPI anchor synthesis (16). Biophysical studies with artificial membranes have shown that very long acyl chain ceramides can coalesce to form ordered domains with unique physical properties (17, 18). These data lead to the hypothesis that C26 ceramide and GPI-AP with C26 ceramide use their physical properties to coalesce into orderly regions or regions in the relatively messy ER membrane lipid environment. It is mainly composed of short and unsaturated glycerolipids (C16:1 and C18:1) (19, 20). These regions will be selectively focused on specific ER exit sites (ERES), where ceramide and ceramide-based GPI-AP can be co-transported to the Golgi in the same dedicated COPII vesicle (5).
In this study, we have directly tested this lipid-based mechanism by using super-resolution confocal real-time imaging microscopy (SCLIM), which is a cutting-edge microscopy technique that can simultaneously observe fluorescently labeled proteins The three-color and three-dimensional (3D) images have extremely high resolution and speed in living cells (21, 22).
We first applied SCLIM technology to further define how normal GPI-AP with C26 ceramide group was screened from transmembrane secreted proteins after leaving the ER in S. cerevisiae. In order to check the classification of ER, we used a genetic system that can directly visualize newly synthesized cargo entering ERES in vivo (7, 23). As cargo, we chose C26 ceramide-based GPI-AP Gas1 labeled with green fluorescent protein (GFP) and transmembrane secreted protein Mid2 labeled with near-infrared fluorescent protein (iRFP), both of which target the plasma membrane (24–26). In the sec31-1 temperature-sensitive mutant, these two cargoes are expressed under a galactose-inducible promoter and a constitutive ERES marker. At the extreme temperature (37°C), because the sec31-1 mutation affects the function of COPII coat component Sec31 to inhibit COPII germination and ER export, newly synthesized cargo accumulates at the ER (23). After cooling to a low temperature (24°C), the sec31-1 mutant cells recovered from the secretory area, and the accumulated new synthetic cargo began to be exported from the ER. CLIM visualization showed that most of the newly synthesized Gas1-GFP and Mid2-iRFP still accumulated in the ER of the sec31-1 mutant cells after incubation at 37°C and then released at 24°C for 5 minutes (Figure 1). Since Mid2-iRFP is distributed on the entire ER membrane, and Gas1-GFP is concentrated and gathered in the discontinuous ER membrane area, their distribution is completely different (Figure 1, A to C and Movie S1). In addition, as shown in Figure 1D, the Gas1-GFP cluster does not have Mid2-iRFP. These results indicate that GPI-AP and transmembrane proteins were separated into different ER membrane regions early. The Gas1-GFP cluster is adjacent to a specific ERES labeled with mCherry’s COPII coat protein Sec13 (Figure 1, E and F, and movie S1) (23).
sec31-1 cells express galactose-induced secretions, a long acyl chain (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, green) and the transmembrane protein Mid2-iRFP (TMP, blue) and this Constructive ERES labeling Sec13-mCherry (ERES, magenta) was incubated at 37°C for 30 minutes, moved to 24°C, and imaged by SCLIM 5 minutes later. (A to C) shows a representative merged or single 2D image of a plane (A), a 2D projection image of 10 z-sections (B) or a 3D cell hemisphere image of cargo and ERES markers (C). Scale bar 1μm (A and B). The scale unit is 0.551μm (C). Gas1-GFP was detected in discrete ER regions or clusters, while Mid2-iRFP was detected and distributed throughout the ER membrane (C). (D) The graph shows the relative fluorescence intensity of Gas1-GFP and Mid2-iRFP in the Gas1-GFP cluster along the white arrow line (left). AU, arbitrary unit. (E and F) represent the 3D image that combines the goods and ERES mark. Gas1-GFP clusters were detected near the specific ERES. The scale unit is 0.551μm. (F) The white solid arrow marks the Gas1-GFP cluster associated with ERES. The middle and right panels show the merged enlarged 3D image and a rotated view of the selected Gas1-GFP cluster.
The close spatial relationship between the Gas1-GFP cluster and a specific ERES indicates that Gas1-GFP can enter selective ERES, which is different from the selectivity used by Mid2-iRFP to leave the ER. To address this possibility, we quantified the ERES ratio for only one or two goods (Figure 2, A to C). We found that most ERES (70%) contain only one type of cargo. The bottom image of Figure 2C shows two typical examples of ERES with only Gas1-GFP (Figure 1) or only Mid2-iRFP (Figure 2). In contrast, approximately 20% of ERES contains two cargoes that overlap in the same area. It was found that some ERES (10%) contained two types of cargo, but they were isolated in clearly different areas. Therefore, this statistical analysis shows that after the ER is exported, GPI-AP Gas1-GFP and the transmembrane cargo Mid2-iRFP are divided into different ERES (Figure 2D). This sorting efficiency is very consistent with the previous biochemical analysis (6) and morphological determination (7). We can also observe the behavior of the quarantined cargo entering ERES (Figure 2E and Movie S2). Figure 2E shows that only a small portion of Gas1-GFP (panel 3) or Mid2-iRFP (panel 4) enters the ERES from one side and is confined in a discrete area. Panel 5 of Figure 2E shows that Gas1-GFP and Mid2-iRFP are sometimes found in the same ERES, but they enter from different sides and are concentrated in separate regions that may represent different COPII vesicles. We also confirmed that the observed separation and classification of C26 ceramide-based GPI-AP Gas1 as selective ERES is specific because another transmembrane secretion cargo, the GFP-tagged plasma membrane protein Axl2 (27 ), showing similar behavior to Mid2-iRFP. (Picture S1 and Movie S3). The newly synthesized Axl2-GFP is distributed through the ER membrane like Mid2-iRFP (Figure S1, A and B), and is co-localized with Mid2-iRFP in most ERES (Figure S1, B to D). Panels 1 and 2 of Figure 1. S1C shows two typical examples of ERES where two transmembrane cargoes overlap. In these cases, both goods enter ERES together (Figure S1E, Panel 3 and Movie S3).
The sec31-1 cells expressing galactose inducible secretions, Gas1-GFP (GPI-AP, green) and Mid2-iRFP (TMP, blue) and constitutive ERES labeling Sec13-mCherry (ERES, magenta) were placed at 37 After incubating for 30 minutes at °C, move to 24 °C to release the secretion block, and image with SCLIM after 20 minutes. (A to C) Representative 2D projection images (A; scale bar, 1μm) or 3D cell hemisphere images (B and C; scale unit, 0.456μm) of the cargo and 10 z-sections marked by ERES. The lower panel in (B) and the panel in (C) display processed images to display only the goods present in ERES (magenta) [Gas1-GFP (gray) and Mid2-iRFP (light blue)]. (C) Open arrow: ERES only carries one piece of cargo (1 to 4). Gray arrow: ERES contains segregated cargo (5). White solid arrow: ERES containing co-located cargo. Below: The selected single ERES contains only Gas1-GFP (1) or Mid2-iRFP (2). Scale bar, 100 nm. (D) Quantification of the photomicrograph described in (C). The average percentage of ERES that contains only one cargo (Gas1-GFP or Mid2-iRFP), segregated cargo and overlapping cargo. In three independent experiments, n=432 in 54 cells. Error bar = SD. Two-tailed unpaired t test. *** P = 0.0002. (E) 3D image of selected ERES of quarantined cargo marked with (C). Gas1-GFP (green) (3) or Mid2-iRFP (blue) (4) enters ERES (magenta) from one side and is restricted to a small area within ERES. Sometimes, both types of cargo enter the same ERES (5) from the same side and are confined to an isolated area within the ERES. Scale bar, 100 nm.
Next, we tested a hypothesis that the long acyl chain ceramide (C26) present in the ER membrane drives the specific clustering and sorting of Gas1 into selective ERES. To this end, we used a modified yeast strain GhLag1, in which the two endogenous ceramide synthases Lag1 and Lac1 were replaced by GhLag1 (the Lag1 homolog of cotton), resulting in a yeast strain with a cell membrane Ceramide strain shorter than wild type (Figure 3A) (28). Mass spectrometry (MS) analysis showed that in wild-type strains, 95% of the total ceramide is very long (C26) chain ceramide, while in GhLag1, 85% of the ceramide is very long (C18 and C16). ), only 2% of ceramide is very long (C26) chain ceramide. Although C18 and C16 ceramides are the main ceramides detected in the GhLag1 membrane so far, MS analysis also confirmed that the GPI anchor of Gas1-GFP expressed in the GhLag1 strain contains C26 ceramide, which is comparable to wild-type lipids. The quality is the same (Fig. 3A) (26). Therefore, this means that the ceramide remodeling enzyme Cwh43 is highly selective for C26 ceramide, as shown in Figure 26, it preferentially incorporates the GPI anchor from a small amount of C26 ceramide in the GhLag1 strain. S2 (29). Nevertheless, the cell membrane of GhLag1 basically only contains C18-C16 ceramide, while Gas1-GFP still has C26 ceramide. This fact makes this strain an ideal tool to specifically solve the problem of the acyl chain length of membrane ceramide in the ER. The hypothetical role of class and sorting. Then, we first studied the ability of C26 Gas1-GFP to accumulate in clusters in GhLag1 with a temperature-sensitive mutant allele of sec31-1 through conventional fluorescence microscopy, where only the long (C18-C16) chain exists in the ER membrane Ceramide (Fig. 3). We observed that in sec31-1, most of Gas1-GFP was concentrated in clusters, while Gas1-GFP in sec31-1 GhLag1 with long (C18-C16) long ceramide ER membrane was mainly not clustered and distributed in In the entire ER membrane. To be precise, because C26 ceramide-based clustering is closely related to specific ERES (Figure 1), we next investigated whether this process may also involve the function of the ER export protein mechanism. GPI-AP uses a special COPII system for ER export, which is actively regulated by Ted1′s structural remodeling of the glycan portion of the GPI anchor (30, 31). The recombinant GPI-glycan is then recognized by the transmembrane cargo receptor p24 complex, which in turn selectively recruits Lst1, which is a specific isoform of the major COPII cargo binding subunit Sec24, forming a GPI-AP-rich COPII Vesicles are necessary (31-33). Therefore, we constructed a double mutant that combined the deletion of these single proteins (the p24 complex component Emp24, GPI-glycan remodeling enzyme Ted1 and the specific COPII subunit Lst1) with the sec31-1 mutant strain, and studied them Is it possible to form Gas1-cluster GFP (Figure 3). We observed that in sec31-1emp24Δ and sec31-1ted1Δ, Gas1-GFP is mainly unclustered and distributed throughout the ER membrane, as previously seen in sec31-1 GhLag1, while in sec31-1lst1Δ, Gas1-GFP Like sec31-1. These results indicate that in addition to the presence of C26 ceramide in the ER membrane, the clustering of Gas1-GFP also needs to bind to the p24 complex, and does not require specific Lst1 recruitment. Then, we explored the possibility that the chain length of ceramide in the ER membrane can regulate the binding of Gas1-GFP to p24. However, we found that the presence of C18-C16 ceramide in the membrane does not affect the GPI-glycans reconstructed by the p24 complex (Figures S3 and S4, A and B) or binding to GPI-AP and exporting GPI-AP. ability. Recruit COPII subtype Lst1 (Figure S4C). Therefore, C26 ceramide-dependent clustering does not require protein interactions with different ER export protein mechanisms, but supports an alternative sorting mechanism driven by lipid length. Then, we analyzed whether the ceramide acyl chain length in the ER membrane is important for the effective classification of Gas1-GFP as selective ERES. Since Gas1 in the GhLag1 strain with short-chain ceramide leaves the ER and enters the plasma membrane (Figure S5), we believe that if the sorting is driven by the length of the ceramide acyl chain, the Gas1 in the GhLag1 strain can be redirected and crossed. ERES goods with the same membrane.
(A) The cell membrane of GhLag1 mainly contains shorter C18-C16 ceramides, while the GPI anchor of Gas1-GFP still has the same C26 IPC as wild-type cells. Above: acyl chain length analysis of ceramide in the cell membrane of wild-type (Wt) and GhLag1p strains by mass spectrometry (MS). The data represents the percentage of total ceramide. The average of three independent experiments. Error bar = SD. Two-tailed unpaired t test. **** P ＜0.0001. Bottom panel: MS analysis of the acyl chain length of the IPC present in the Gas1-GFP (GPI-IPC) GPI anchor expressed in the wild-type and GhLag1p strains. The data represents the percentage of total IPC signal. Average of five independent experiments. Error bar = SD. Two-tailed unpaired t test. ns, not important. P = 0.9134. (B) Fluorescence micrographs of sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ and sec31-1lst1Δ cells expressing galactose-induced Gas1-GFP were incubated at 37°C for 30 minutes and passed down to Perform routine fluorescence microscopy after 24°C. White arrow: ER Gas1-GFP cluster. Open arrow: Unclustered Gas1-GFP is distributed on the entire ER membrane, showing the ER characteristic nuclear ring staining. Scale bar, 5μm. (C) Quantification of the photomicrograph described in (B). The average percentage of cells with punctate Gas1-GFP structure. In three independent experiments, n≥300 cells. Error bar = SD. Two-tailed unpaired t test. **** P ＜0.0001.
To directly solve this problem, we performed SCLIM visualization of Gas1-GFP and Mid2-iRFP in GhLag1 with the sec31-1 temperature-sensitive mutant allele (Figure 4 and Movie S4). After the ER was retained at 37°C and subsequently released at 24°C, most of the newly synthesized Gas1-GFP was not clustered and distributed throughout the ER membrane, as observed by conventional microscopes (Figure 4, A and B ). In addition, a large percentage of ERES (67%) includes two types of cargo co-located in it (Figure 4D). Panels 1 and 2 of Figure 4C show two typical examples of ERES with overlapping Gas1-GFP and Mid2-GFP. In addition, both goods were recruited into the same ERES (Figure 4E, panel 3 and movie S4). Therefore, our results indicate that the length of the ceramide acyl chain in the ER membrane is an important determinant of ER protein aggregation and classification.
Sec31-1 GhLag1 cells expressing galactose-induced secretions, Gas1-GFP (GPI-AP, green) and Mid2-iRFP (TMP, blue) and constitutive ERES-labeled Sec13-mCherry (ERES, magenta) Incubate at 37°C. Continue for 30 minutes, drop to 24°C to release secretions, and image with SCLIM after 20 minutes. (A to C) Representative 2D projection images (A; scale bar, 1μm) or 3D cell hemisphere images (B and C; scale unit, 0.45μm) of the 10 z-sections marked by cargo and ERES. The lower panel in (B) and the panel in (C) display processed images to display only the goods present in ERES (magenta) [Gas1-GFP (gray) and Mid2-iRFP (light blue)]. (C) White filled arrow: ERES, goods overlap. Open arrow: ERES contains only one item. Lower panel: The selected ERES has overlapping goods (1 and 2) marked in (C). Scale bar, 100 nm. (D) Quantification of the photomicrograph described in (C). In the sec31-1 and sec31-1 GhLag1 units, only one cargo (Gas1-GFP or Mid2-iRFP) is included, and the average percentage of ERES for isolated cargo and overlapping cargo. In three independent experiments, n = 432 in 54 cells (sec31-1) and n = 430 in 47 cells (sec31-1 GhLag1). Error bar = SD. Two-tailed unpaired t test. *** P = 0.0002 (sec31-1) and ** P = 0.0031 (sec31-1 GhLag1). (E) 3D image of selected ERES with overlapping cargo (3) marked in (C). Gas1-GFP (green) and Mid2-iRFP (blue) approach ERES (magenta) from the same side and stay in the same ERES restricted area. Scale bar, 100 nm.
This study provides direct in vivo evidence that lipid-based protein cargoes are classified into selective export sites in the secretory pathway, and reveals the importance of acyl chain length for classification selectivity. Using a powerful and cutting-edge microscopy technique called SCLIM, we demonstrated the newly synthesized Gas1-GFP (a major plasma membrane GPI-AP with a very long acyl chain (C26) ceramide lipid portion) in yeast ) The regions clustered in discrete ERs are associated with specific ERES, while transmembrane secreted proteins are distributed throughout the ER membrane (Figure 1). In addition, these two types of goods enter different ERES selectively (Figure 2). The acyl chain length of the cellular ceramide in the membrane is reduced from C26 to C18-C16, the Gas1-GFP cluster is disrupted into the discrete ER region, and Gas1-GFP is rerouted to leave the ER with the transmembrane protein through the same ERES (Figure 3 and Figure 3). 4).
Although GPI-AP uses a specialized protein mechanism to exit ER, we found that C26 ceramide-dependent separation does not rely on differential protein interactions that may lead to ERES specialization (Figures S4 and S5). Instead, our findings support an alternative classification mechanism driven by lipid-based protein clustering and subsequent exclusion of other cargoes. Our observations indicate that the Gas1-GFP region or cluster associated with a specific ERES lacks the transmembrane secreted protein Mid2-iRFP, which indicates that the C26 ceramide-dependent GPI-AP cluster will facilitate their entry into the relevant ERES, and at the same time, exclude transmembrane The secretions enter this particular ERES (Figures 1 and 2). In contrast, the presence of C18-C16 ceramides in the ER membrane does not cause GPI-AP to form regions or clusters, so they do not exclude or replace transmembrane secreted proteins into the same ERES (Figures 3 and 4). . Therefore, we propose that C26 ceramide drives separation and classification by facilitating the clustering of proteins linked to specific ERES.
How to achieve this C26 ceramide-dependent clustering into a specific ER area? The tendency of membrane ceramide to separate laterally may cause GPI-AP and C26 ceramide to form small and instantaneously ordered lipids in the more irregular lipid environment of the ER membrane containing shorter and unsaturated glycerolipids. Quality clusters (17, 18). These small temporary clusters can be further fused into larger, more stable clusters after binding to the p24 complex (34). Consistent with this, we showed that C26 Gas1-GFP needs to interact with the p24 complex to form larger visible clusters (Figure 3). The p24 complex is a heterozygous oligomer composed of four different p24 transmembrane proteins in yeast (35), which provides multivalent binding, which can lead to cross-linking of small GPI-AP clusters, thereby generating larger Stable cluster (34). The interaction between the protein ectodomains of GPI-APs may also contribute to their aggregation, as shown during their Golgi transport in mammalian polarized epithelial cells (36). However, when C18-C16 ceramide is present in the ER membrane, when the p24 complex binds to Gas1-GFP, large separate clusters will not be formed. The underlying mechanism may depend on the specific physical and chemical properties of the long acyl chain ceramide. Biophysical studies of artificial membranes show that although both long (C24) and short (C18-C16) acyl chain ceramides can cause phase separation, only long acyl chain ceramides (C24) can promote high Curvature and film bending to reshape the film. Through mutual reference (17, 37, 38). It has been shown that the transmembrane helix of TMED2, the human homologue of Emp24, selectively interacts with C18 ceramide-based sphingomyelin in the cytoplasmic lobules (39). Using molecular dynamics (MD) simulations, we found that both C18 and C26 ceramides accumulate around the cytoplasmic lobules of the Emp24 transmembrane helix, and they have similar preferences (Figure S6). It is worth noting that this indicates that the transmembrane helix of Emp24 can lead to asymmetric distribution of lipids in the membrane. This is a recent result based on mammalian cells. Similar MD simulations also show the presence of ether lipids (40) . Therefore, we speculate that C26 ceramide in the two lobules of ER26 is locally enriched. When GPI-AP in the luminal lobules directly binds to multivalent p24 and the accumulation of C26 ceramide around p24 in the cytoplasmic lobules, it can promote the accompanying Protein aggregation and membrane curvature are generated through the fingers (41), causing GPI-AP to separate into discrete regions adjacent to ERES, which also favors the highly curved regions of the ER membrane (42). Previous reports supported the proposed mechanism (43, 44). The multivalent binding of oligolectins, pathogens or antibodies to ceramide-based glycosphingolipids (GSL) on the plasma membrane triggers large GSL aggregation, enhances phase separation and causes membrane deformation and internalization (44). Iwabuchi etc. (43) It was found that in the presence of long (C24) but not short (C16) acyl chains, the multivalent ligand bound to GSL lactosylceramide induced the formation of large clusters and membrane invagination, and the cytoplasm Lyn-mediated signal transduction on the leaflets is interdigitated by acyl chains in coupled neutrophils.
In mammalian polarized epithelial cells, the concentration of the anti-Golgi network (TGN) to the level of the apical plasma membrane controls the separation and sorting of GPI-AP (10, 45). This aggregation is driven by GPI-AP oligomerization (36), but it may also depend on the ceramide chain length we find in yeast. Although mammalian GPI-AP has an ether lipid-based anchor, and its chemical structure is very different from the very long acyl chain ceramide, a recent study found that both lipids have evolutionarily similar physical and chemical properties And function (40). Therefore, the ether lipid part in mammalian cells may be similar to the C26 ceramide in yeast, and its role is to associate with the long-chain ceramide in the membrane to promote GPI-AP aggregation and sorting. Although this possibility still needs to be tested directly, previous findings support that the transport of the long acyl chain ceramide to the Golgi body is not carried out by cytoplasmic transfer proteins, but depends on the synthesis of GPI anchors like yeast. Therefore, the evolutionary conservative mechanism seems to be able to selectively co-transport very long acyl chain ceramide and GPI-AP (13, 16, 20, 46, 47) in the same transport vesicle.
In yeast and mammalian polarized epithelial cell systems, GPI-AP aggregation and separation from other plasma membrane proteins all occur before reaching the cell surface. Paladino et al. (48) found that on the TGN of mammalian polarized epithelial cells, GPI-AP clustering is not only necessary for the selective classification of GPI-APs to the apical plasma membrane, but also regulates the clustering organization of GPI-APs and Its biological activity. Cell surface. In yeast, this study showed that the C26 ceramide-dependent GPI-AP cluster on the ER can regulate the cluster organization and functional activity of GPI-AP on the plasma membrane (24, 49). Consistent with this model, GhLag1 cells are allergic to GPI inhibitors or drugs that affect cell wall integrity (28), and the need for functional Gas1-GFP clusters (49) of the tip ceramide projected in the mating of yeast cells indicates G Possible physiological consequences of hLag1 cells. GPI-AP error. However, further testing whether the functional organization of the cell surface has been programmed from the ER by a sorting method based on lipid length will be the subject of our future research.
The Saccharomyces cerevisiae strains used in this work are listed in Table S1. The MMY1583 and MMY1635 strains of SCLIM for live cell imaging were constructed in the background of W303. These strains expressing Sec13-mCherry with a fluorescent protein tag were constructed using a polymerase chain reaction (PCR)-based method with pFA6a plasmid as a template (23). The strain expressing Mid2-iRFP labeled with fluorescent protein under the control of the GAL1 promoter was constructed as follows. PCR amplification of iRFP-KanMx sequence from pKTiRFP-KAN vector (gift of E. O’Shea, Addgene plasmid number 64687; http://n2t.net/addgene: 64687; research resource identifier (RRID): Addgene_64687) And inserted into the C-terminus of endogenous Mid2. After the Mid2-iRFP genome sequence was amplified and cloned into the GAL1 promoter, it was integrated into the Not I-Sac I site of the integration plasmid pRS306. The resulting plasmid pRGS7 was linearized with Pst I to integrate into the URA3 locus.
The Gas1-GFP fusion gene is expressed under the control of the GAL1 promoter in the centromere (CEN) plasmid, which is constructed as follows. The Gas1-GFP sequence was amplified by PCR from the pRS416-GAS1-GFP plasmid (24) (gift of L. Popolo) and cloned into the Xma I–Xho I site of the CEN plasmid pBEVY-GL LEU2 (gift of C) . Miller; Addgene plasmid number 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). The resulting plasmid was named pRGS6. The Axl2-GFP fusion gene is also expressed under the control of the GAL1 promoter of the pBEVY-GL LEU2 vector, and its construction is as follows. The Axl2-GFP sequence was amplified from pRS304-p2HSE-Axl2-GFP plasmid (23) by PCR, and cloned into the Bam HI-Pst I site of pBEVY-GL LEU2 vector. The resulting plasmid was named pRGS12. The sequence of the oligonucleotides used in this study is listed in Table S2.
The strain was supplemented with 0.2% adenine and 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1 % Yeast extract and 2% protein ept). (YPR)] or 2% galactose [YP-galactose (YPG)] as a carbon source, or in a synthetic minimal medium (0.15% yeast nitrogen base and 0.5% ammonium sulfate) to supplement appropriate amino acids and bases required for nutrition , And containing 2% glucose (synthetic glucose minimal medium) or 2% galactose (synthetic galactose minimal medium) as a carbon source.
For real-time imaging, temperature-sensitive sec31-1 mutant cells expressing the construct under the GAL1 promoter were grown in YPR medium at 24°C overnight to mid-log phase. After induction in YPG at 24°C for 1 hour, the cells were incubated in SG at 37°C for 30 minutes, and then transferred to 24°C to release from the secretion block. Concanavalin A was used to fix the cells on a glass slide and imaged by SCLIM. SCLIM is a combination of Olympus IX-71 inverted fluorescence microscope and UPlanSApo 100×1.4 numerical aperture oil lens (Olympus), high-speed and high-signal-to-noise ratio rotating disc confocal scanner (Yokogawa Electric), custom spectrometer, and custom cooling The system’s image intensifier (Hamamatsu Photonics) can provide a magnifying lens system with a final magnification of ×266.7 and a charge-coupled device camera that multiplies electrons (Hamamatsu Photonics) (21). Image acquisition is performed by custom software (Yokogawa Electric). For 3D images, we used a custom-made piezoelectric actuator to vibrate the objective lens vertically, and collected the optical parts 100 nm apart in a stack. The Z-stack image is converted into 3D voxel data, and the theoretical point spread function used for the rotating disc confocal microscope is used for deconvolution processing by Volocity software (PerkinElmer). By using Volocity software to automatically threshold for co-location analysis, ERES including cargo was measured. Line scan analysis was performed using MetaMorph software (Molecular Devices).
Use GraphPad Prism software to determine statistical significance. For the two-tailed Student’s t-test and the ordinary one-way analysis of variance (ANOVA) test, differences between groups are considered to have a significant impact on P <0.05 (*).
For fluorescence microscopy of Gas1-GFP, the log phase cells were grown overnight in YPD and collected by centrifugation, washed twice with phosphate buffered saline, and incubated on ice for at least 15 minutes, and then proceeded under the microscope as previously described Check (24). The Leica DMi8 microscope (HCX PL APO 1003/1.40 oil PH3 CS) equipped with an objective lens, L5 (GFP) filter, Hamamatsu camera and Application Suite X (LAS X) software was used for acquisition. .
The samples were denatured with SDS sample buffer at 65°C for 10 minutes, and then separated by SDS-polyacrylamide gel electrophoresis (PAGE). For immunoblotting analysis, 10 μl of sample was loaded per lane. Primary antibody: Use rabbit polyclonal anti-Gas1 at a dilution of 1:3000, rabbit polyclonal anti-Emp24 at a dilution of 1:500, and rabbit polyclonal anti-GFP (a gift from H. Riezman) at a dilution of 1:3000. The mouse monoclonal anti-Pgk1 antibody was used at a dilution of 1:5000 (a gift from J. de la Cruz). Secondary antibody: Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) used at a dilution of 1:3000 (Pierce). HRP-conjugated goat anti-mouse IgG was used at a dilution of 1:3000 (Pierce). The immune response zone was observed by the chemiluminescence method of SuperSignal West Pico reagent (Thermo Fisher Scientific).
As described in (31), a natural immunoprecipitation experiment was performed on the enriched ER fraction. In short, wash yeast cells with TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor mixture) at 600 nm ( OD600) at 100 optical density twice. It was broken with glass beads, and then the cell debris and glass beads were removed by centrifugation. The supernatant was then centrifuged at 17,000 g for 15 minutes at 4°C. The pellet was resuspended in TNE and digitalis saponin was added to a final concentration of 1%. The suspension was incubated for 1 hour with rotation at 4°C, and then the insoluble components were removed by centrifugation at 13,000 g at 4°C for 60 minutes. For Gas1-GFP immunoprecipitation, first pre-incubate the sample with empty agarose beads (ChromoTek) at 4°C for 1 hour, and then incubate with GFP-Trap_A (ChromoTek) at 4°C for 3 hours. The immunoprecipitated beads were washed five times with TNE containing 0.2% digoxigenin, eluted with SDS sample buffer, separated on SDS-PAGE, and analyzed by immunoblotting.
As described in (31), cross-linking determination was performed on the enriched ER fraction. Briefly, the enriched ER fraction was incubated with 0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, 20 min). The crosslinking reaction was quenched by adding glycine (50 mM final concentration, 5 minutes, 20°C).
As previously described (50), MS analysis of ceramide in wild-type and GhLag1 strains was performed. In short, cells were grown to exponential phase (3 to 4 OD600 units/ml) in YPD at 30°C, and 25×107 cells were harvested. Their metabolism is quenched with trichloroacetic acid. Use extraction solvent [ethanol, water, ether, pyridine and 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] and 1.2 nmol of internal standard C17 ceramide (860517, Avanti polar lipid) quality). Use monomethylamine reagent [methanol, water, n-butanol and methylamine solution (4:3:1:5 v/v)] to perform mild alkaline hydrolysis of the extract, and then use water-saturated n-butanol to desalt. Finally, the extract was resuspended in a positive mode solvent [chloroform/methanol/water (2:7:1) + 5 mM ammonium acetate] and injected into the mass spectrometer. Multi-reaction monitoring (MRM) was performed for the identification and quantification of sphingolipid molecules. The TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) is equipped with a robotic nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) for lipid analysis. The collision energy is optimized for each ceramide category. MS data was obtained in positive mode. For each biological replicate, the lipid signal is the median of three independent measurements.
As described in (31), the cells (800×107) expressing Gas1-GFP were subjected to natural immunoprecipitation. The purified Gas1-GFP was separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The protein was visualized by staining PVDF with amide black. The Gas1-GFP band was cut from the PVDF and washed 5 times with methanol and once with liquid chromatography-MS (LC-MS) grade water. By incubating the membrane strip with 500μl 0.3 M NaOAc (pH 4.0), buffer and 500μl freshly dissolved 1 M sodium nitrite mixture at 37°C for 3 hours, the lipid fraction is released from Gas1-GFP and lysed Release of inosine phosphate ceramide between glucosamine and inositol (51). After that, the membrane strip was washed four times with LC-MS grade water, dried at room temperature, and stored in a nitrogen atmosphere at -80°C until analysis. As a control, a blank sample of PVDF membrane was used for each experiment. The lipid extracted from Gas1-GFP was then analyzed by MS as described (50). In short, PVDF strips containing GPI-lipid were resuspended in 75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] and passed electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid species (TSQ Vantage). In this case, MS data was obtained in negative ion mode.
As mentioned earlier, the lipid portion of the GPI anchor was separated from the [3H]-inositol-labeled GPI-AP (16). The lipids were separated by thin-layer chromatography using a solvent system (55:45:10 chloroform-methanol-0.25% KCl) and visualized using FLA-7000 (Fujifilm).
The cells expressing Gas1-GFP (600×107) were washed twice with TNE buffer with TNE buffer, and broken with glass beads, and then centrifuged to remove cell debris and glass beads. The supernatant was then centrifuged at 17,000 g for 1 hour at 4°C. The pellet was washed in TNE and incubated with 1 U PI-PLC (Invitrogen) in TNE containing 0.2% digitalis saponin for 1 hour at 37°C. After the enzyme treatment, the membrane was removed by centrifugation at 17,000 g at 4°C for 1 hour. To immunoprecipitate Gas1-GFP, the supernatant was incubated with GFP-Trap_A (ChromoTek) at 4°C overnight. The purified Gas1-GFP separated by SDS-PAGE was stained with Coomassie brilliant blue. The Gas1-GFP staining band was cut off from the gray surrounding the aqueduct, and then after alkylation with iodoacetamide and reduction with dithiothreitol, in-gel digestion with trypsin was performed. Extract and dry tryptic peptides and peptides with GPI-glycans. The dried peptide was dissolved in 20 μl of water. Inject a portion (8μl) into the LC. An octadecylsilane (ODS) column (Develosil 300ODS-HG-5; inner diameter 150 mm×1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) was used to separate peptides under specific gradient conditions. The mobile phase is solvent A (0.08% formic acid) and solvent B (0.15% formic acid in 80% acetonitrile). An Accela HPLC system (Thermo Fisher Scientific, Boston, Massachusetts) was used to elute the column with solvent A within 55 minutes at a flow rate of 50 μl min-1 for 5 minutes, and then the concentration of solvent B was increased to 40%. , United States). The eluate was continuously introduced into the ESI ion source, and the tryptic peptides and peptides with GPI-glycans were analyzed by LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). In the MS setup, the voltage of the capillary source was set to 4.5 kV, and the temperature of the transfer capillary was kept at 300°C. The capillary voltage and tube lens voltage were set to 15 V and 50 V, respectively. MS data was obtained in the positive ion mode (resolution of 60,000; mass accuracy of 10 parts per million) in a mass range of 300/m/z mass/charge ratio (m/z) 3000. The MS/MS data is obtained through the ion trap in the LTQ Orbitrap XL [the first 3 digits on which the data depends, collision induced dissociation (CID)].
MD simulations were performed using GROMACS (52) software and MARTINI 2 force field (53-55). The CHARMM GUI Membrane Builder (56, 57) was then used to construct a bilayer containing dioleoylphosphatidylcholine (DOPC) and Cer C18 or DOPC and Cer C26. The topology and coordinates of Cer C26 are derived from DXCE by removing the extra beads from the sphingosine tail. Use the process described below to balance the double layer and run it, then use the last coordinates of the system to build a system containing Emp24. The transmembrane domain of yeast Emp24 (residues 173 to 193) was constructed as an α-helix using the visual MD (VMD) tool molecular structure (58). Then, after removing the overlapping lipids, the protein was coarsely granulated and inserted into the bilayer using CHARMM GUI. The final system contains 1202 DOPC and 302 Cer C26 or 1197 DOPC and 295 Cer C18 and Emp24. Ionize the system to a concentration of 0.150M. Four independent replicates were made for two bilayer compositions.
The lipid bilayer is balanced using the CHARMM GUI process, which involves minimizing and then balancing 405,000 steps, where the position constraints are gradually reduced and eliminated, and the time step is increased from 0.005 ps to 0.02 ps. After equilibration, it produces 6 µs with a time step of 0.02 ps. After inserting Emp24, use the same CHARMM GUI process to minimize and balance the system, and then run for 8 s in production.
For all systems, during the balancing process, the pressure is controlled by the Berendsen barostat (59), and during the production process, the pressure is controlled by the Parrinello-Rahman barostat (60). In all cases, the average pressure is 1 bar and a semi-isotropic pressure coupling scheme is used. In the balance and production process, a thermostat (61) with speed recalibration is used to couple the temperature of protein, lipid and solvent particles respectively. During the entire operation, the target temperature is 310K. The non-bonding interaction is calculated by generating a pairing list using the Verlet scheme with 0.005 buffer tolerance. The Coulomb term is calculated using the reaction field and a cut-off distance of 1.1 nm. The Vander Waals term uses a cut-off scheme with a cut-off distance of 1.1 nm, and the Verlet cut-off scheme is used for potential drift (62).
Using VMD, the cutoff wavelength between DOPC phosphate beads or ceramide AM1 beads and the protein is 0.7 nm, and the number of lipids that interact with the protein is calculated. According to the following formula, calculate the depletion-enrichment (DE) factor as in (63): DE factor = (the amount of total lipids in the protein 0.7) in the protein 0.7 (the amount of Cer in total lipids)
The reported value is obtained as an average, and the error bars are four independent copies of SE. The statistical significance of DE factor is calculated by t test [(averageDE-factor-1)/SE]. Calculate the P value from the one-tailed distribution.
The GROMACS tool was used to calculate the 2D lateral density map of the system containing Emp24 within the last 250 ns of the trace. In order to obtain the enrichment/depletion map of ceramide, the density map of Cer is divided by the sum of the map of Cer and DOPC, and then divided by the concentration of Cer in the body. The same color map scale is used.
For supplementary materials for this article, please see http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
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