Spatial partitioning of secretory cargo from Golgi resident proteins in live cells
© White et al; licensee BioMed Central Ltd. 2001
Received: 4 August 2001
Accepted: 10 October 2001
Published: 10 October 2001
To maintain organelle integrity, resident proteins must segregate from itinerant cargo during secretory transport. However, Golgi resident enzymes must have intimate access to secretory cargo in order to carry out glycosylation reactions. The amount of cargo and associated membrane may be significant compared to the amount of Golgi membrane and resident protein, but upon Golgi exit, cargo and resident are efficiently sorted. How this occurs in live cells is not known.
We observed partitioning of the fluorescent Golgi resident T2-CFP and fluorescent cargo proteins VSVG3-YFP or VSVG3-SP-YFP upon Golgi exit after a synchronous pulse of cargo was released from the ER. Golgi elements remained stable in overall size, shape and relative position as cargo emptied. Cargo segregated from resident rapidly by blebbing into micron-sized domains that contained little or no detectable resident protein and that appeared to be continuous with the parent Golgi element. Post-Golgi transport carriers (TCs) exited repeatedly from these domains. Alternatively, entire cargo domains exited Golgi elements, forming large TCs that fused directly with the plasma membrane. However, domain formation did not appear to be an absolute prerequisite for TC exit, since TCs also exited directly from Golgi elements in the absence of large domains. Quantitative cargo-specific photobleaching experiments revealed transfer of cargo between Golgi regions, but no discrete intra-Golgi TCs were observed.
Our results establish domain formation via rapid lateral partitioning as a general cellular strategy for segregating different transmembrane proteins along the secretory pathway and provide a framework for consideration of molecular mechanisms of secretory transport.
During secretory transport, organelle resident proteins must separate from itinerant secretory cargo. In the Golgi apparatus, resident glycosylation enzymes sequentially modify secretory proteins after delivery from their site of synthesis in the endoplasmic reticulum (ER). Different classes of cargo are then sorted in the Golgi and delivered to specific final destinations – an intracellular compartment or the cell exterior – leaving the resident proteins behind. The Golgi itself comprises a collection of stacked membrane cisternae with a distinct architecture . Golgi resident glycosylation enzymes are type II (N-terminus in the cytosol) transmembrane proteins with the catalytic domain extending into the Golgi lumen. Their localization information is contained in the transmembrane and closely adjacent regions, but consensus Golgi targeting motifs have not been defined [2–4]. Golgi resident-GFP (green fluorescent protein) fusion proteins diffuse rapidly within interconnected cisternal membranes, but remain tightly localized to Golgi stacks . Transmembrane secretory cargo also diffuses laterally within Golgi membranes , but with diffusion constants several-fold lower than those measured for Golgi residents [5, 6]. Golgi glycosylation enzymes must have intimate access to itinerant cargo to in order to carry out covalent modification reactions, and so cargo and enzyme must be mixed within Golgi membranes for at least the time required to carry out the reaction. Nevertheless, rapidly-diffusing Golgi resident and cargo are so efficiently sorted after mixing that Golgi glycosylation enzymes are not detectable at the cell surface or in post-Golgi intracellular compartments.
The flux of transmembrane cargo through the Golgi can be high enough that the amount of cargo and associated membrane traversing the Golgi is significant compared to the amount of Golgi membrane . When a synchronous pulse of secretory transport is visualized using a GFP fusion to the glycoprotein of the ts045 mutant of vesicular stomatitis virus (VSVG-GFP), the wave of material arriving at the cell surface from the Golgi is enough to cause visible expansion of the plasma membrane . Biochemically, it is obvious that the addition of a relatively large amount of material to the Golgi should dilute out its enzymatic and transport machinery, which would be expected to alter the efficiency of transport. However, detailed, quantitative analysis in live cells shows transport efficiency, indicated by kinetic rate constants, remains unaltered as cargo empties from the Golgi . Morphologically, it might be expected that Golgi elements should greatly expand, then shrink as they absorb, then disgorge a wave of cargo , but the effect of a pulse of cargo transport on Golgi morphology in live cells has not yet been described. It is therefore unclear how the content and structure of the Golgi is maintained against relatively high levels of cargo during a pulse of secretory transport [8–10].
In addition to the sorting of resident transmembrane proteins from cargo, different classes of transmembrane cargo are segregated from one another in the late Golgi or trans-Golgi network (TGN). Targeted delivery of different classes of cargo contributes to the generation and maintenance of overall cellular polarity . Recent work has addressed how different classes of cargo separate from one another in the Golgi in live cells . Proteins of the "apical" or "basolateral" classes of cargo are preferentially delivered to the apical or basolateral membrane of polarized cells and contain sorting signals which direct targeted delivery [11, 13]. During exit from the Golgi, these sorting signals manifest themselves at the cellular level (even in non-polarized cells [12, 14, 15]) by organizing domains – distinct regions of apparently continuous membrane – that contain exclusively one class of cargo or the other . These domains are large enough to be visualized in live cells, and their generation occurs prior to or concomitant with the generation of cargo-specific transport carriers, which then translocate to the plasma membrane and fuse to deliver cargo to the cell surface . Previously, we visualized cargo with Golgi or TGN resident proteins to pinpoint the precise cellular location of apical and basolateral cargo separation . Here, we focus in more detail on how cargo segregates from transmembrane Golgi resident proteins. We note similarities in the dynamics of segregation of cargo from Golgi residents and the dynamics of apical/basolateral cargo segregation. Our observations have implications for the molecular mechanisms underlying segregation of cargo and Golgi glycosylation enzymes during transit through as well as exit from the Golgi.
Despite the high flux of exiting transmembrane cargo, Golgi elements remained stable in overall size, shape, and position (Figure 1B) and Additional filesets 1. Visually, cargo fluorescence in the Golgi became progressively fainter as post-Golgi TCs exited (Figure 2A). Quantitation of relative fluorescence levels showed that this was due transfer of fluorescence from the Golgi region to the plasma membrane (Figure 2B). The decrease of cargo in the Golgi during this time was approximately linear. Previous work demonstrated that cargo levels in the Golgi rise and fall in a nonlinear manner over the full course of cargo transport (ER→ Golgi→ PM, ). However, there is a period where the decrease exhibits linear behavior (Figure 2B, inset), corresponding to the period of maximal Golgi exit , so it is reasonable that we observe a linear decrease of cargo levels in the Golgi at these times. Regardless of exact exit kinetics, Golgi elements remained intact after cargo emptied from them (see Additional Filesets 1 and 2), maintaining their overall size, shape and position whether they contained cargo or not. Thus, Golgi elements appear relatively unaltered by the passage of a pulse of cargo.
where A is a constant, would show a linear relation between concentration and time. Importantly, recovery after bleaching Golgi resident (instead of cargo) lacks the linear component – there is only an exponential recovery process which plateaus . We did not observe discrete transport intermediates trafficking into the bleached region, but we did occasionally observe tubular connections containing cargo between closely juxtaposed Golgi elements Additional Filesets 3, 4, 5, 6 and 7. In PtK2 cells, the Golgi consists of separate elements scattered in the MTOC region. We never observed cargo or resident transport between these elements (Additional Filesets 3, 4, 5, 6 and 7.) Consistent with this, we saw no recovery when an entire separate element was bleached, regardless of whether cargo or resident fluorescence was bleached (our unpublished observations). Thus, intra-Golgi transport intermediates do not traverse long distances (between separated Golgi elements); this is clear from our results but inconsistent with published cell fusion experiments [26, 27]. Recovery within interconnected Golgi elements could be mediated by intermediates too small to be imaged, or continuous with Golgi membranes, consistent with EM observations [1, 28]. Regardless, our observation of cargo transfer between Golgi elements shows that photobleaching experiments may be able to monitor intra-Golgi transport.
The partitioning of cargo from resident Golgi protein appears to be a distinct step from the generation of post-Golgi transport carriers. Although TCs tend to exit from cargo domains, suggesting that cargo domains concentrate the molecular machinery required for formation of post-Golgi TCs, the formation of a large domain does not seem to be a prerequisite for TC exit, since we observe TCs exiting from regions where there is no notable cargo domain. One possibility is that in some cases, TC exit too rapidly to allow the accumulation of visible levels of cargo in domains. Regardless, the two events – domain formation and TC exit – are not closely coupled.
Since the Golgi resident T2-CFP is restricted to Golgi stacks , it is not clear whether domain formation represents transfer of cargo from the stacks to TGN regions (as previously proposed ), or whether it represents the last step prior to formation of a true post-Golgi TC. The observation that entire domains exit the Golgi, translocate outward, and fuse directly with the plasma membrane (Figure 5), indicates that cargo domains have a significant degree of post-Golgi TC "character," that is, they have most of the machinery of a post-Golgi TC. Additionally, we observed previously that cargo domains form on elements labelled with the TGN-resident protein TGN38-YFP , which makes it seem likely that at least some of the domains observed with T2-CFP as the resident marker are true cargo domains, in the sense that they contain no resident protein. Indeed, no integral membrane residents of post-Golgi TCs have yet been definitively identified. Our observations most likely show a range of molecular events, from transfer into TGN regions to the generation of large post-Golgi TCs which have not yet detached. If so, it is notable that spatial partitioning of resident from cargo occurs in the same manner (lateral segregation), regardless of the type of structure that is being generated (TGN element or post-Golgi TC).
Our observations raise several important points for models of transport through the Golgi. Golgi elements are dynamic, moving and changing gradually over time, but their dynamics to not observably change during the passage of a pulse of cargo, and they remain relatively stable on this time scale. In contrast, cargo distribution changes rapidly within Golgi elements; cargo domains form and the distribution of cargo becomes polarized as transport progresses . Together, these observations indicate that cargo passes through a pre-existing, relatively stable Golgi structure rather than one that is continuously generated by coalescence of cargo [29–31]. The Golgi elements visualized here most likely correspond to a complete Golgi stack, including the full complement of cis-, medial-, and trans- cisternae. Thus, our observations are most consistent with the passage of cargo cis to trans through pre-existing, stably maintained sets of stacked cisternae. This view of the Golgi as a stable entity is consistent with the existence of Golgi structural and scaffolding proteins [32–38].
Additionally and more directly, it appears that Golgi elements are structurally stable during intra-Golgi transport, since Golgi elements labelled by T2-CFP maintained their size and shape during the bleach experiment (Figure 7 and Additional Fileset 7, compare pre-bleach with recovery), and we believe that our photobleaching experiments detect transport of cargo within Golgi elements, amongst or between stable cisternae (Figure 7). Notably, recovery of cargo appears to be confined to continuously or interconnected Golgi elements, since we failed to observe exchange of cargo between obviously separate, discrete elements, even those in close proximity (within a micron). Thus, we posit that cargo transport is restricted to Golgi cisternae in the same stack, in contrast to the cell fusion studies [26, 27] that are the basis for in vitro assays of intra-Golgi transport [39, 40]. Since different Golgi cisternae can be distinguished at the level of light microscopy , a feasible future experimental direction would be use triple-labeling with three variants of fluorescent protein to directly observe movement of cargo between cisternae.
It is clear that cargo and resident must segregate within the Golgi stack , as well as upon Golgi exit. Thus it is reasonable to propose that the same general mechanism which partitions cargo and resident upon Golgi exit – lateral segregation – may also serve to partition cargo from resident within the stack, albeit with different molecular machinery. Novel isoforms of COPI coatomer subunits , localized to Golgi cisternae (as opposed to Golgi-associated vesicles), could meet the functional criteria (Figure 8) – binding of cytosolic signal sequences [43, 44], and rapid polymerization – and so may be good candidates to partition cargo and resident by lateral segregation mechanisms (as in Figure 8). If Golgi cisternae are interconnected, even if only transiently [1, 28, 45], partitioning of cargo into domains could drive rapid movement of cargo between discrete cisternae. It seems necessary to propose an additional mode of transport within the Golgi because current models , which present the Golgi as an "iterative sorting device," gradually filtering cargo from resident, fail to explain several features: the lack of dilution effects when high levels of cargo are pulsed through the Golgi , the explosive disassembly of the Golgi upon BFA treatment , and the rapid, formation of cargo domains by lateral segregation (observed here and ).
Materials and Methods
Constructs, tissue culture, cell lines, have been described previously [12, 19]. Live cell laser-scanning confocal microscopy and calibration of the instrumentation was performed as in . All movies were taken in line-interlace mode (switching C/YFP channels between lines) to allow colocalization of moving elements .
For Figure 2, fluorescence was quantitated by measuring average pixel intensities in the entire cell, in the Golgi region, and a background region outside of the cell. Background pixel intensities were subtracted from the raw measurements, and fluorescence normalized to the initial fluorescence intensities in the region (such that the average initial value was 1). Relative fluorescence intensity was then plotted against time.
Sizes of fluorescent structures in this paper were interpreted with the following properties of light microscopy in mind. For a complete discussion, see . Assuming the lateral resolution of our microscope configuration is 200 nm, spherical objects smaller than 200 nm will appear 200 nm in size. Spherical objects larger than 200 nm will have an apparent diameter that is the sum of their actual diameter plus 200 nm. Thus, a bead with a diameter of 500 nm will have a diameter of about 700 nm. The apparent size of an object is independent of its fluorescence intensity unless the signal is saturated. If the signal is saturated, the center part of the bright spot will appear to be larger than it actually is. Note that the size ratio of two similarly saturated objects will be the same as the unsaturated ratio, meaning that relative comparisons can still be made even in the presence of saturation. The movies in this paper have been contrast enhanced for clear presentation, the original data are unsaturated.
Bleaching experiments were carried out starting 45 minutes after shift from 40°C to 32°C in the presence of 100 μg/ ml of cycloheximide, added upon shift to 32°C, to inhibit new protein synthesis. Cells were imaged for approximately 10 minutes prior to bleaching under normal imaging conditions. Then a region encompassing several Golgi elements was bleached in the YFP channel only using a 514 nm Ar-Kr laser line at full power. Control experiments showed bleaching in the YFP channel did not effect fluorescence in the CFP channel. Recovery was monitored by automatically resuming the pre-bleach imaging conditions and acquiring an image every 6 to 10 s. It was impossible to monitor full recovery because cargo was continuously exiting the Golgi. Images shown in the movie are enhanced for display and so may contain saturated pixels, but the raw images were unsaturated.
Recovery was quantitated in both channels by measuring the average pixel intensities in the bleached and unbleached region after first subtracting average background pixel intensities. For both channels the ratio of bleached:unbleached was calculated, and this ratio normalized so that the average pre-bleach value of the region was 1. These values were used to normalize for fluctuations in focus and cell movement by taking the ratio of the YFP signal to CFP (cargo to resident). This ratio was plotted against time for Figure 7.
cyan fluorescent protein
confocal laser-scanning microscope/microscopy
green fluorescent protein
spacer (15 aa insert)
vesicular stomatitis virus glycoprotein
yellow fluorescent protein.
We gratefully thank the CCC development team: Stephan Albrecht (DSP programming), Alfons Riedinger (software design and programming), Georg Ritter (digital hardware), Nick Salmon (software design and programming), Thomas Stefany (optics), and Reiner Stricker (analog hardware). We thank Tom Rapoport and Anne Hart for their support in the final stages of this work and Melissa Rolls for comments on the manuscript.
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