The GM130 and GRASP65 Golgi Proteins

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NATURE CELL BIOLOGY VOL 3 DECEMBER 2001 http://cellbio.nature.com 1101

The GM130 and GRASP65 Golgi proteinscycle through and define a subdomain

of the intermediate compartmentPierfrancesco Marra*, Tania Maffucci*, Tiziana Daniele*, Giuseppe Di Tullio*, Yukio Ikehara,Edward K. L. Chan, Alberto Luini*, Gala Beznoussenko*, Alexander Mironov* &

Maria Antonietta De Matteis*

*Department of Cell Biology and Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy

Department of Biochemistry, Fukuoka University School of Medicine, Fukuoka 814-0180, Japan

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA

e-mail: [email protected]

Integrating the pleomorphic membranes of the intermediate compartment (IC) into the array of Golgi cisternae is acrucial step in membrane transport, but it is poorly understood. To gain insight into this step , we investigated thedynamics by which cis-Golgi matrix proteins such as GM130 and GRASP65 associate with, and incorporate, incom-

ing IC elements. We found that GM130 and GRASP65 cycle via membranous tubules between the Golgi complex anda constellation of mobile structures that we call late IC stations. These stations are intermediate between the IC andthe cis-Golgi in terms of composition, and they receive cargo from earlier IC elements and deliver it to the Golgicomplex. Late IC elements are transient in nature and sensitive to fixatives; they are seen in only a fraction of fixedcells, whereas they are always visible in living cells. Finally, late IC stations undergo homotypic fusion and establishtubular connections between themselves and the Golgi. Overall, these features indicate that late IC stations mediatethe transition between IC elements and the cis-Golgi face.

The Golgi apparatus has a complex architecture composed ofstacks of flat cisternae connected in a ribbon-like fashion bytubular reticular areas. How such an organization is achieved

at the cis-Golgi face and maintained through the Golgi in spite ofthe continuous flux of membranes remains a central problem in

the biology of transport. Two classes of protein complexes arethought to be involved in morphogenesis of the Golgi complex,possibly through a coordinated action: the Golgi spectrinactinskeleton and the Golgi matrix proteins. The spectrin skeletonassembles on Golgi membranes in a fashion dependent on theactivity of the small GTPase ARF (ADP-ribosylation factor) and onthe level of phosphatidylinositol 4,5-bisphosphate1,2. The spectrinskeleton facilitates incorporation of endoplasmic reticulum (ER)-derived membranes into the cis-Golgi1; it may also control thestructure of Golgi stacks, on the basis of the probable similarity ofthe known role of spectrin at the plasma membrane3. The Golgimatrix proteins4,5 were originally identified as a set of insolubleGolgi proteins, and include GM130 and GRASP65. These proteinsare involved in postmitotic reassembly and stacking of the Golgicisternae68 and can assemble in a structure reminiscent of the

Golgi even in the absence of Golgi resident proteins suggesting thatthey can act as primary scaffold components underpinning theGolgi architecture9. Remarkably, GM130, GRASP65 and otherGolgi matrix proteins are located primarily at the cis-pole of theGolgi stacks4,6, where they probably function in the incorporationof the ER-derived membranes into the Golgi.

The membranes that reach the cis-Golgi are tubular clusters thatconstitute the ERGolgi intermediate compartment (IC)10. Thebiogenesis, maintainance, and identity of the IC have been and areobjects of intense study. Current theories suggest that the IC may beconsidered either as an outgrowth of the ER11,12, as a part of the cis-Golgi network (CGN)13, or as a separate compartment1417. In the lat-ter case, the IC might be viewed as a stable compartment15,16 or as atransient compartment made up of transport intermediates operat-ing between the ER and the Golgi14,17. The difficulties in reconciling

these apparently contrasting views and in defining the boundary ofthe IC result largely from the disperse distribution and pleomor-phic organization of this compartment, and from its dynamicbehaviour10. Even the molecular composition of the IC elements ishighly heterogenous18 and includes proteins continuously cycling

between the ER and the Golgi complex.However, by combining thebroadest definition of the ERGolgi IC (consisting of the mem-branes interposed between the ER and the Golgi stacks) with theinformation derived from the dynamics of proteins traversing thiscompartment (which includes cargo proteins19,20, recycling pro-teins21, and the coat complexes COPI and COPII2224), it is possibleto distinguish three layers in the IC. The first layer includes the ERexit sites (ERES, or transitional ER), marked by COPII, and madeup of rather stationary elements22,24. The second layer consists oftubular clusters that move long distances, travelling in centripetaldirection along microtubules; this layer contains COPI, but notCOPII, and forms what we define from here onwards as IC. Thethird layer includes the CGN,which consists of tubular clusters jux-taposed to the cis-Golgi cisterna (which in turn contain the cis-Golgi markers GM130, GRASP65, mannosidase I, and Helix

Pomatia (HP) lectin-binding proteins25). According to the view ofthe IC as a transient compartment, these three layers correspond tothree stages of the IC membrane lifespan: origin at the ERES via asorting process mediated by the SAR1pCOPII complex; motor-driven translocation towards the Golgi along microtubules andconcomitant loss of ER-recycled components; and final incorpora-tion into the Golgi complex.Although the first two stages have beenextensively studied and their molecular machineries satisfactorilyelucidated, the process by which the IC membranes are incorporat-ed into the cis-Golgi remains elusive.

To gain insight into the details of this final step, we investigatedhow Golgi-structural components such as the matrix proteinsGM130 and GRASP65 associate with the incoming IC membranes.We find that IC elements acquire these Golgi components beforecontacting the cis-pole of the central Golgi area, at the level of a

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specialized subdomain of the IC that we define as late IC (L-IC);this is where the transition between IC and cis-Golgi takes place.

ResultsGM130 cycles between the Golgi complex and peripheral trans-port stations via membranous tubules. We examined the dynamicsof GM130 in living cells by analysing a GFPGM130 chimaera inCOS7 fibroblast and NRK epithelium cells. This chimaeric proteinbehaves exactly like the endogenous GM130 under all the condi-tions tested (see Methods), and becomes incorporated into theGolgi matrix to the same extent as GM130 (data not shown). Whenimaged in living COS7 cells, GFPGM130 localized not only at thecentral Golgi area, as expected4, but also on two types of peripher-al structures, which we define as puncta and tubules (Fig. 1A). TheGFPGM130-containing puncta were distributed throughout the

cytoplasm but were more concentrated around the Golgi area, hadan average apparent size of around 0.8m (ranging from 0.3 to 3

m), and were highly mobile. They moved with speeds rangingfrom 0.2 to 0.8 m s1 and a directionality that suggests micro-tubule-based motility, that is, towards the central Golgi or otherpuncta, which they often eventually joined. While moving, theyreceived and emitted tubules. The tubules were more transient andmobile than the puncta. They elongated and moved in centrifugal,centripetal and lateral directions. The shape, distribution andmovement patterns of puncta and tubules were highly complex.Sometimes the tubules originated from GFPGM130-labelledperipheral puncta, projected centripetally towards the Golgi areawhile still connected to the original puncta, and after reaching thisarea, disappeared into the Golgi mass (Fig. 1B and C; andSupplementary Information movies 1 and 2). Other tubulesextended centrifugally from the central area towards the peripheral

NATURE CELL BIOLOGY VOL 3 DECEMBER 2001 http://cellbio.nature.com1102

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Figure 1 GM130 cycles between the Golgi complex and peripheral compart-

ments via tubules. A, Distribution of GFPGM130 in COS7 cells. Note that

GFPGM130 localizes not only to the central Golgi area, but also to peripheral

puncta and tubules. BF, Dynamics of GFPGM130 in living COS7 cells. Time is

given in min.sec. B, Snapshots from Supplementary Information movie 1. An antero-

grade tubule originating from a puncta (01.18) elongates towards the Golgi area

(01.20), transiently connects the donor and acceptor compartments (01.21),

detaches from the puncta (01.22), and finally disappears into the central Golgi area

(01.24) while the peripheral puncta remains apparently unchanged compared with

the first frame (compare 01.18 with 01.24). C, Snapshots from Supplementary

Information movie 2. Frames from 00.09 to 00.30 show a retrograde tubule origi-

nating from the Golgi area (asterisk), detaching from the latter, moving towards,

and fusing with a peripheral puncta (indicated with a filled arrowhead). The trajecto-

ry of the movement of the puncta is indicated with a dashed line. The same puncta,

receives a tubule from a neighbouring puncta (00.34). The tubule transiently con-

nects the two puncta before fusing and disappearing into the acceptor puncta

(00.37). D, GM130 retrograde tubules mediate the fluorescence recovery after pho-

tobleaching (FRAP) of the peripheral puncta. The peripheral area included between

the two red profiles and including puncta and tubules was bleached (one bleaching

round of 100 iterations, frame 00.00) and the FRAP of this area was recorded over

2 min after the bleaching. Note that numerous tubules emanate from the Golgi area

(1.08, 1.31), generate puncta that are interconnected by tubules (1.56) and finally

appear as separate puncta (1.58). E, Bleaching of the Golgi area induces fluores-

cence loss of the GM130-positive puncta. Five bleaching rounds (total time, 3 min)

of the Golgi area (indicated by the red profile in a) induced almost complete loss of

fluorescence in the puncta (b). F, Bleaching of the puncta and tubules induces fluo-

rescence loss of the Golgi. The peripheral area, delimited by the red profile ( a and

b), and then the area including puncta and tubules and delimited by the blue profile

(a and d), were repeatedly bleached (five bleaching rounds over 16 and 10 min,

respectively), and the fluorescence of the Golgi area was measured in c and e,

respectively. It is evident that the bleaching of the blue area ( d, e), but not that of

the red area (b, c) reduced the fluorescence in the Golgi area (compare e with c).

Scale bars:A, 4 m; B, 2 m; C, 4 m; D, 8 m; E, 6 m; F, 6 m.



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puncta. The retrograde direction of these tubules was verifiedstudying the fluorescence recovery after photobleaching (FRAP) ofthe peripheral puncta: the fluorescence recovery of the bleached

puncta was clearly mediated by long tubules originating from thecentral Golgi and extending towards the periphery (Fig. 1D). Othertubules were seen to emanate from peripheral puncta, elongatetowards neighbouring or distanct puncta, thus connecting periph-eral puncta with each other, and finally disappear into the acceptorstructures. In some cases the tubules did not establish continuitybetween two structures, but apparently detached from the punctafrom which they originated and then moved towards their destina-tion (Fig. 1C and Supplementary Information movie 2).The averagetubule life span was 5 s (ranging from 2 to 11 s), and length was 6m(ranging from 3 to 12 m). The tubules were collinear with micro-tubules, and were dependent on the presence of an intact micro-tubule system, as they disappeared in cells treated with nocodazole(data not shown). Thus, both the tubules and the puncta are mobile,but tubules are more transient and dynamic; in fact, puncta behave

as slowly moving platforms that emit tubules. Overall, the complexdynamics of these elements suggest that tubules create a permanentsemistable system of interconnections between peripheral puncta

and between puncta and the GM130 central membrane pool. Theresult is a spatially dispersed, but dynamically well connected,membranous system.

To investigate the dynamic relationships between the peripheraland central pools of GM130, the Golgi area or the puncta andtubules were bleached repeatedly, after which the GFPGM130-positive structures were examined using the FLIP (fluorescence lossin photobleaching) technique. Five bleaching rounds (total time 3min) of the Golgi area induced almost complete loss of fluores-cence in the puncta (Fig. 1E), and five bleaching rounds (total time10 min) of the puncta and tubules significantly decreased the fluo-rescence in the Golgi area by 70% (Fig. 1F, d and e). These dataindicate that the pools of GM130 located at the Golgi and atperipheral puncta communicate rapidly. To determine whether anotherwise undetectable cytosolic and/or ER pool might be involved

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GM130

GM130

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GM130

ERGIC53

SEC31 Merge

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Mann l GiantinMann llHP

GRASP65 COPSEC13p115KDEL-R

Figure 2 GM130-positive peripheral structures have compositional features

of an IC subdomain. A, GM130 does not colocalize with cis- or medial-Golgi

markers on puncta and tubules. NRK cells (a, b, e, f), RBL cells (c, d) and COS7

cells (g, h) were labelled with anti-GM130 antibodies (a, c, e, g) and with anti-man-nosidase I (b), anti-mannosidase II (f), anti-giantin (h) antibodies, or with FITC-conju-

gated HP lectin (d). Note that the GM130-positive puncta and tubules (filled arrow-

heads) are not labelled by anti-mannosidase I or II, anti-giantin antibodies, or HP

lectin (empty arrowheads). B, C, GM130-positive tubules and puncta identify a sub-

domain of the IC. B, GM130 colocalizes with GRASP65 and IC markers, but not

with ERES markers on puncta and tubules. COS7 cells were double stained with

anti-GM130 (a, c, e, g, i) and with anti-GRASP65 (b), anti KDEL-R (d), p115

(f), SEC13 (h) or COP (j) antibodies. Note that the GM130-positive puncta and

tubules (filled arrowheads) contain GRASP65 and markers of the IC (KDEL, p115,

COP), but almost exclude SEC13 (a marker of the ERES, empty arrowheads).

Note also the IC markers and SEC13 are present on many peripheral puncta (some

of which are indicated by filled arrows) that are devoid of GM130 (empty arrows).

C, COS7 cells were transfected with GFPGM130 (green) and then processed forimmunofluorescence, and labelled for an ERES marker (SEC31, blue) and for an IC

marker (ERGIC53, red). The boxed area is enlarged in the insets. Four populations

of elements can be distinguished: one labelled exclusively by SEC31 (blue), one by

SEC31 and ERGIC53 (purple), one labelled exclusively by ERGIC53 (red), and one

by ERGIC53 and GM130 (yellow). The last population corresponds to the IC subdo-

main identified by GM130 (L-IC), whereas the red elements represent the E-IC (see

text). Note that in many cases, the overlap of the stainings of SEC31 and ERGIC53

is not complete and the blue (SEC31) and red (ERGIC53) elements are not concen-

tric. Scale bars, 4 m.



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in GM130 dynamics, an extensive cytoplasmic area including theER, but devoid of recognizable GFPGM130-positive structures,was bleached repeatedly (five bleaching rounds in 16 min): underthese conditions no FLIP of the Golgi area was observed (Fig. 1F, band c), indicating that a cytosolic pool and/or ER pool of GM130does not contribute to these fast dynamic processes, and that theobserved anterograde and retrograde tubules are responsible forthe communication between the central and peripheral GM130compartments. Cells that overexpress GFPGM130 (which we usu-ally excluded from our analysis) contain a large pool of cytosolicGFPGM130. In these cells, bleaching of cytoplasmic areas devoidof GM130-positive puncta and tubules induced FLIP in the Golgiarea. Given that in normal cells there is very little cytosolic GM130(ref. 4), we assume that the cytosolic distribution of overexpressedGFPGM130 results from saturation of the membrane-bindingsites (that is, GRASP65). Our results indicate that there is a fastGolgiIC recycling pathway for GM130; they do not, however,exclude the possibility that a slower recycling pathway involving theER might exist and be relevant for longer-term processes.

The peripheral GM130 tubules and puncta seem to undergo sig-nificant changes in abundance over time, even in individual cells.We observed single cells at 4-h intervals and registered the rate ofpuncta and tubule formation over 12 h. This rate was found to vary

on average from 0.3 to 10 tubules per minute. Given the correlationbetween the number of GM130-positive tubules and the transportof cargo proteins (see below and Table 1), the oscillations in theabundance of puncta and tubules might reflect an undetected cyclicactivity of the secretory pathway, possibly related to stages of thecell cycle.GM130-positive peripheral structures have compositional fea-tures intermediate between those of the cis-Golgi and the IC. Ourobservations of GM130 distribution in living cells prompted us tore-examine the distribution of endogenous GM130 and its colocal-ization with Golgi and IC markers by immunofluorescence. Thesensitivity of the tubules to the fixatives was a technical difficulty.When cells expressing GFPGM130 were fixed with paraformalde-hyde (v/v 4%), most tubules were either fragmented into strings ofsmall punctate structures, or became extremely thin or unde-

tectable. Nevertheless, there was a sufficient number of structuresto enable us to characterize the compartment. Consistent with theobservations in live cells, GM130 was localized at the central Golgicomplex, at peripheral puncta, and also at the extremely thintubules that connect the puncta with each other and with the cen-tral Golgi area (Fig. 2). Both the puncta and the tubules labelledwith GM130 were devoid ofbona fideGolgi markers such as giantin(Fig. 2A), the cis-Golgi markers mannosidase I and HP lectin-bind-ing proteins25 (see below and Fig. 3), and the medial-Golgi markermannosidase II. In contrast, there was very good colocalization ofGM130 and GRASP65 under all experimental conditions (Fig. 2B),indicating that these two proteins are closely associated in theperipheral as well as in the central Golgi pool. The peripheralGM130-positive structures were detectable in different cell lines,including COS7, NRK, RBL (Fig. 2), HepG2 and HeLa cells (data

not shown), although always in a fraction of the cell population(~30 10%; this estimate might be low, however, because of thetransient nature of the GM130-labelled tubules and puncta andtheir sensitivity to fixatives).

Some of the features of the GM130-positive puncta and tubulessuggest a similarity with the IC10,21,26,27. To investigate, we analysedthe distribution of GM130 compared with that of IC proteins, suchas COPI, COP, ERGIC5326, KDEL-receptor (KDEL-R), p115 (Fig.2B) and p23. In all cases GM130 and the IC markers colocalize(91 2% of GM130 puncta contain KDEL-R, and 78 2% ofGM130 puncta contain COP), but the distribution of the ICmarkers is broader than that of GM130 (12 4% of KDEL-R-pos-itive and 5 2% ofCOP-positive structures contain GM130). Thepartitioning between the Golgi and peripheral pool was differentfor the IC markers and GM130: about 80% (77 15%) of total

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Figure 3 Ultrastructural analysis of GM130-positive tubules and puncta

with correlative light-electron microscopy (CLEM) and cryo-immuno-

electromicroscopy (cryo-EM). A, B, Cells grown on cover slips with coordi-

nated grids were fixed and labelled for GM130 inA, and for GM130 and COP

in B. The cells exhibiting long GM130-positive tubular protrusions were select-

ed and optical sections performed to determine the spatial positions of the

tubules. Then cells were immunogold-labelled for GM130, embedded and

100 nm-tangential serial sections were examined without additional contrast.

Aa, Low-magnification EM image of the GM130-labelled area identified by

LSCM (inset). Arrowheads show reference points along the nuclear membrane.

Arrows indicate the Golgi area and a tubule labelled for GM130. The rectangu-lar box includes the GM130-positive tubule and the asterisk inside the rectan-

gle indicates a lipid droplet. b, Enlargement of the rectangular box in a, show-

ing the straight tangential membrane tubule (thin arrows) containing GM130

and closely associated with a lipid droplet (asterisk). The breaks along the

tubule represent its bending, which is visible in consecutive sections (data not

shown). c, d, Images from the same series demonstrating the GM130 localiza-

tion at peripheral tubular clusters (c) and at the Golgi (d). c shows a GM130-

positive tubular cluster localized in an area far from the Golgi. Arrowheads indi-

cate ER. d shows the concentration of GM130 labelling at the cis-side of the

stack. B, COS7 cells were immunofluorescence-labelled for GM130 and COP.

The merge of the stainings is shown in c; the single labellings of GM130 and

COP are shown in a and b, respectively. d shows a low-magnification EM

image of the GM130-labelled area identified by LSCM in a. The arrow in ad

indicates a tubule that is positive for GM130, and also, though in a disconti-

nous manner, for COP. e shows a larger view of box in d, including the tubularstructure. Note that the tubular structure identified by immunofluorescence cor-

responds to a bundle of tubules with some of them containing flattened

domains. C, COS7 cells were labelled with HRP-conjugated HP lectin and anti-

GM130 antibodies. GM130 and HP lectin colocalize and show a polarized dis-

tribution in the first two cisternae. The swelling of the cisterna is very likely

due to an over-development of the diaminobenzidine reaction used for HRP

detection. DF, COS7 cells were processed for cryo-EM and labelled for

GM130 (10 nm gold particles, arrows) and mannosidase I (5 nm gold particles,

arrowheads). GM130 and mannosidase I colocalize at the level of the first (or

first two) cisterna of the Golgi stacks (D and E). Colocalization is not seen on

peripheral tubular clusters that have GM130 but lack mannosidase I (E and F).

Scale bars:Aa, 650 nm and inset, 2.6 m; b, 140 nm; c, 225 nm; d, 160 nm;

Bac , 20 m; d, 4 m; e, 300 nm; C, 220 nm; D, 200 nm; E, 260 nm; F,

120 nm.



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GM130 labelling but only 40% (41 12%) of total COPI labellingcoincided with the central Golgi area. All the non-Golgi GM130labelling fell in the next 10 m around the Golgi area (the averagediameter of COS7 cells is around 30 m), whereas only 60% of thenon-Golgi COPI labelling was present in the same area, with therest distributed in more peripheral zones. Also, the average size ofCOPI elements was smaller than those of GM130 puncta (theapparent average diameter of COPI-positive and GM130-negativeelements was 0.25 m and of COPI- and GM130-positive elementswas 0.6 m). We also analysed the extent of colocalization ofGM130 with the IC markers on the tubules. We found that it washigher (80 7%) in the case of proteins such as KDEL-R and p23that cycle between the Golgi and the ER than in the case of thosesuch as ERGIC53 that cycle mostly in peripheral compartments

(that is, between the IC and the ER (30 7%)). COPI and p115were not uniformly distributed along the GM130-positive tubules,but were organized in spots collinear with the tubules (Fig. 2B).

These data indicate that GM130 delineates a subdomain of theIC, which includes the larger IC elements that are distributed in awide area (with an average radius of 10 m) around the Golgi com-plex, and communicate with it via anterograde and retrogradetubules.

There was poor colocalization between GM130 and proteinslocalized at the ERES, such as the COPII components SEC13 orSEC31 (less than 10% of GM130 puncta colocalized with SEC31;Fig. 2B). There was only a partial overlap between the ERES mark-ers and the IC markers (KDEL-R): 17 4% of KDEL-positive ele-ments apparently colocalized with SEC31 and 16 5% of SEC31

VSV-G

GM130

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Figure 4 GM130-positive L-IC elements contain and transport secretory

cargo. COS7 cells were transfected with GFPVSV-G, kept 16 h at 40 C, shifted

to 32 C for the indicated times, fixed and processed for immunofluorescence. At

40 C VSV-G is localized in the ER (data not shown).A, The cells were stained with

anti-GM130 antibodies. Three minutes after the shift to 32 C (a, b), VSV-G accumu-

lates in punctate structures (filled arrows in a), which are devoid of GM130 (empty

arrows in b), whereas at 8 min after the temperature shift VSV-G colocalizes with

GM130 on puncta and tubules (filled arrows in c and d). Forty minutes after the

temperature shift (e, f), VSV-G localizes to the Golgi, the plasma membrane, and

peripheral spots (filled arrows) that are devoid of GM130 (empty arrows) and proba-

bly represent post-Golgi transport intermediates. B, GFPVSV-G-transfected cells

were double stained for KDEL-R (red) and SEC31 (blue) in a and b and for KDEL-R

(red) and GM130 (blue) in c and d. The insets show a larger view of the boxed

areas in ad, and includes the single labelling and the merge of the labelling as indi-

cated. Three minutes after the shift to 32 C (a, c), VSV-G accumulates in punctate

structures that contain SEC31 (light blue spots in a) or KDEL-R (yellow spots in a

and c), but that are devoid of GM130 (blue puncta in c). Note that only few VSV-G

containing puncta are positive both for SEC31 and KDEL-R (white spots in a). Eight

minutes after the shift to 32 C (b, d), there are still some VSV-G containing puncta

positive for KDEL-R but devoid of GM130 or SEC31 (yellow spots in b and d) but

also VSV-G containing puncta positive both for KDEL and GM130 (white spots and

tubule in d). Note that at this time only few VSV-G puncta contain SEC31 (light blue

puncta in b). Scale bars,A, 10 m; B, 4 m.



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elements colocalized with KDEL. It is worth noting that the stain-ings of SEC31 and KDEL confirmed previous observations22, indi-cating that the labelled structures might correspond to subdomainsof the same structure overlapped.

Finally, in triple-labelling experiments, it was possible to followthe distribution of an ERES marker (SEC31), an IC marker(ERGIC53) and GM130 in the same cell (Fig. 2C). Without takinginto account the central Golgi area where, due to the resolution lim-its of the confocal analysis, it was impossible to distinguish specific

staining patterns, four main populations were identified: onelabelled exclusively by SEC31 (blue spots in Fig. 2C), one by SEC31and ERGIC53 (purple spots, which often show a local segregationof red and blue staining), one labelled exclusively by ERGIC53 (redspots), and one, representing the GM130-positive subdomain ofthe IC, labelled by ERGIC53 and GM130 (yellow spots andtubules).Ultrastructure of the peripheral IC elements containing GM130.We next characterized the ultrastructure of the puncta and bonafide tubules positive for GM130 using correlative light-electronmicroscopy (CLEM28,29). This technique allows the ultrastructure ofindividual cells and organelles previously identified at theimmunofluorescence level to be analysed (Fig. 3A and B). In thecentral Golgi area, GM130 was mainly restricted to the cisface ofthe Golgi4, where it localized to the first two cisternae of the stacksand colocalized with the HP lectin25 (Fig. 3C). The GM130 labelwas present also on a variety of complex membranes comprising:flat reticulated cisterna-like structures underlying the first cisternaof the stacks; tubulo-reticular elements apparently connectingadjacent stacks (presumably belonging to the noncompact zone);and clusters of convoluted tubular profiles (with an average diam-eter of 800 nm). The tubular clusters were identified as typical ICelements after analysis of serial sections. These labelled IC mem-

branes were located not only close to the Golgi area but also in areasdistant from the Golgi (Fig. 3Ac). These peripheral elements corre-sponded to the large GM130-positive puncta observed in immuno-fluorescence experiments (see above). GM130 antibodies alsolabelled long and continous membranous tubules (defined as tubu-lar profiles visible in no more than two consecutive 100-nm-thickserial sections) emanating from GM130-stained tubular clusters.These tubules were 50100 nm thick and 312 m long (Fig. 3Aand B). Usually the tubules were straight or slightly bent. Strikingly,many were arranged in pairs or in bundles with some of them con-taining flattened domains (Fig. 3B), and in these cases the tubulescorresponded at the immunofluorescence level to thick tubularstructures containing both GM130 and COPI. Using cryoimmuno-electron microscopy (cryo-EM), we found that mannosidase I colo-calized with GM130 on the cis-Golgi cisterna, but was not present

on the peripheral tubular clusters containing GM130 (Fig. 3DF).The EM results described above were consistent with observa-tions both in living and in fixed cells, and showed that GM130 asso-ciates not only with the cis-Golgi cisternae and tubular clustersclose to the Golgi stacks, but also with peripheral straight tubulesand tubular clusters containing IC markers and devoid ofcis-Golgimarkers.GM130-positive IC elements carry secretory cargo. To assesswhether the IC elements that contain GM130 are involved in trans-porting cargo, the distribution of GM130 was studied simultane-ously with that of a secretory membrane protein, the temperature-sensitive variant of the glycoprotein (G) from the ts045 strain ofthe vesicular stomatitis virus (VSV). Transport of the VSV-G pro-tein from the ER to the Golgi can be synchronized because it foldsincorrectly and is retained in the ER at 40 C; correct folding and

release occurs after shifting to 32 C. Cells were transfected withGFPVSV-G, kept 16 h at 40 C to accumulate the VSV-G in theER, shifted to 32 C for different time intervals, and then processedfor immunofluorescence and stained for GM130 (Fig. 4A) or forSEC31 and KDEL-R or for KDEL-R and GM130 (Fig. 4B). Threeminutes after the shift to 32 C VSV-G accumulates in punctatestructures devoid of GM130, and at 8 min VSV-G colocalizes withGM130 on puncta and tubules (Fig. 4A). The punctate structuresthat contain VSV-G at early times (3 min) show very good colocal-ization with the ERES marker SEC31 (58% of VSV-G positive ele-ments contained SEC31) and with the IC markers KDEL-R (87% ofVSV-G positive elements contained KDEL-R). We were able to dis-tinguish a population of VSV-G-containing structures positive forIC markers, but not for SEC31 (Fig. 4B, a). At these times(23 min) the colocalization between VSV-G and GM130 was poor

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Figure 5 GM130-positive L-IC elements transport secretory cargo.

a, b, Dynamics of VSV-G transport to and from the GM130-positive tubules and L-IC

stations. COS7 cells doubly transfected with CFPVSV-G (green) and YFPGM130

(red) were kept overnight at 40 C and then shifted to 32 C. a and b show individ-

uals frames of movies taken 1015 min after release from the 40 C block. a, A

GM130-positive puncta (L-IC, filled arrowhead) initially devoid of VSV-G (empty

arrowhead), acquires VSV-G (00.04), and generates/transforms into a tubule

(00.06). Afterwards, the tubule reaches (00.28) and disappears into the central

Golgi area (00.32). b, A GM130- and VSV-G-positive tubule (filled arrowhead) origi-

nates from a GM130- and VSV-G-positive puncta (00.00), detaches from the puncta

(00.02), moves (00.11, 00.17), then reaches and fuses with a neighbouring puncta

(00.21). Scale bars, 2 m. c, VSV-infected COS7 cells were kept for 2 h at 40 C

and then shifted to 32 C for 8 min. The cells were processed for cryo-EM and the

sections were double stained for GM130 (5 nm gold particles, arrowheads) andVSV-G (10 nm gold particles, arrows). Note that GM130 and VSV-G colocalize at

the cis-cisterna and at vesicular and tubular profiles juxtaposed to the cis-cisternae.

Scale bar, 70 nm.



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(less than 10% of GM130-positive puncta contained VSV-G;Fig. 4A, d, and B, c). After 510 min the VSV-G became associatedwith larger spots and long tubules connecting the spots to eachother and to the central Golgi area. At this stage, many (85 10%)

GM130-positive structures were positive for VSV-G (Fig. 4A and B,d). At later times (15 min), VSV-G concentrated into the centralGolgi area,and 3040 min after the temperature shift, VSV-G local-ized on both puncta and tubules (probably representing post-Golgitransport intermediates), which were not labelled for either ICmarkers or GM130, and on the plasma membrane (Fig. 4A, c andf).

We define as early-IC (E-IC) elements that receive cargo soon(23 min) after warming to 32 C. E-IC are mainly peripheral andcontain IC markers but are devoid of COPII and GM130 (yellowspots in Fig. 4B, c and d).We define as late-IC elements that receivecargo 510 min after warming to 32 C. These elements are lessperipheral than the E-IC, and contain both GM130 and IC mark-ers (white spots in Fig. 4B, d), but are devoid ofcis-Golgi markers(such as mannosidase I or HP; Figs 1A and 3E).

We examined the progression of VSV-G through the pre-Golgitransport segment using time-lapse imaging in living cells expressingCFPVSV-G and YFPGM130 (Fig. 5A). After 16 h of incubation at40 C, VSV-G was found in the ER, whereas GM130 was associated

with the Golgi complex, as well as with peripheral puncta andtubules. After shifting to 32 C, VSV-G progressively associated withpunctate structures that moved centripetally in a stop-and-go fash-ion, as described previously19,20. Consistent with the immunofluores-cence data reported above, the extent of colocalization betweenGM130 and VSV-G was low when the cargo protein was in the smallpunctate peripheral structures (that is, ERES and E-IC elements),and increased when the cargo reached the larger and more centralelements of the IC (that is, L-IC elements). Indeed, many of thepuncta that contained GM130 were initially devoid of VSV-G, butacquired it later. The transfer of cargo to the L-IC units occurredmainly by direct contact between VSV-G-containing E-IC elementsand GM130-positive tubules and/or puncta. After cargo receipt, theGM130 puncta behaved dynamically in several ways. Some trans-formed into tubules that extended and moved to the central Golgi,

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Figure 6 GM130 distribution after synchronization at 15 C. A, VSV-infected

COS7 cells (ac) were kept for 2 h at 40 C and then shifted for an additional 2 h

at 15 C. The cells were double stained with anti-GM130 (a) and anti-VSV-G (b) anti-

bodies; the merged pattern is shown in c. Note that the more central and larger

puncta positive for VSV-G also contain GM130 (filled arrowheads), whereas the

more peripheral puncta contain VSV-G (filled arrows) but are devoid of GM130

(empty arrows). COS7 cells (df,jl) or NRK cells (gi) were incubated for 2 h at

15 C, processed for immunofluorescence and then double stained for GM130 (d,g,j) and ERGIC53 (e), KDEL-R (h) or SEC13 (k). The merges of the two staining

patterns are shown in f, i, and l. Note that GM130 partially redistributes to periph-

eral puncta, there colocalizing with markers of the IC (ERGIC53, KDEL-R, filled

arrowheads). However, the IC markers are also present on more peripheral spots

(filled arrows) devoid of GM130 (empty arrows). Note that in contrast to what is

observed under steady-state conditions shown in Fig. 2, there is a marginal colocal-

ization between GM130 and SEC-13 on punctate structures (filled arrowheads); see

text for details. mo show the transport of GM130 after release from the 15 C

block. VSV-infected COS7 cells, were kept for 2 h at 40 C, for an additional 2 h at

15 C and then shifted to 32 C for 3 min. The cells were fixed, processed for

immunofluorescence and stained for GM130 and VSV-G. Note that a large number

of tubules containing GM130 and VSV-G (filled arrowheads) connect the peripheral

puncta with the central Golgi area. B, Dynamics of YFPGM130 and CFP-VSV-G

upon release from the 15 C block. COS7 cells double-transfected with

YFPGM130 and CFPVSV-G were kept overnight at 40 C, for additional 2 h at15 C and then shifted at 32 C and imaged. After 4 min at 32 C (00.30) the

central Golgi area (dotted line) was bleached (00.00) by 200 iterations at maximal

laser-beam intensity and the cells were imaged for an additional 2.5 min. Note that

many tubules containing VSV-G and GM130 (filled arrowheads) move towards the

central Golgi area, and that the latter gradually reacquires both VSV-G and GM130-

fluorescence. Scale bars, 10 m.



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and then disappeared into it (Fig. 5A). Sometimes cargo-contain-ing tubules detached from the GM130- and VSV-G-positive L-ICstations, moved towards the Golgi area or other GM130-positiveelements, and finally fused with them (Fig. 5B). Less frequently,GM130-positive tubules emanated from the L-IC stations andestablished stable (or repetitive) connections between these stationsand the Golgi; in these cases, VSV-G-positive spots could be seenmoving along/within the GM130-positive tubules. Finally, thecargo and GM130-containing L-IC stations established multiple,variably transient tubular connections with other stations and with

the central Golgi area and underwent homotypic fusion. Theseobservations indicate that the GM130-positive L-IC receives theER-derived cargo from earlier IC elements and delivers it to theGolgi complex via anterograde tubules. An apparent discrepancywith this conclusion was that in a few cases the cargo appeared toarrive directly at the central Golgi area (inside a GM130-negative E-IC element), apparently bypassing the GM130-positive L-IC andtubules. However, and as is shown by the EM data in Fig. 3, thereare numerous GM130-containing tubular clusters in the centralGolgi area. These exceptions might, therefore, be due to the resolu-tion limits of the LSCM analysis, which allows the arrival of cargo-containing E-IC elements at the L-IC stations to be detected onlywhen these stations are sufficiently far from the Golgi complex(and not when the L-IC stations are very close to, and not resolv-able from, the central Golgi). We therefore used cryo-EM studies to

examine VSV-infected cells: these studies showed a high degree ofcolocalization of VSV-G with GM130 at the level of central tubularclusters (Fig. 5C). The same studies showed that not all the periph-eral tubular clusters containing VSV-G contained GM130, con-firming the observation that the GM130-positive tubular clustersidentify a subdomain of the IC.

Finally, we studied the dynamics of GM130-positive L-IC andtubules under conditions in which the ERGolgi membrane trans-port is arrested at the IC by a prolonged exposure to 15 C (refs 10,17,21, 26,27; Fig. 6).After 2 h at 15 C, the IC of VSV-infected cells

was filled with VSV-G and, consistent with previously publishedreports30, was enlarged compared with that of noninfected cells.Under these conditions, the association of GM130 with the IC wasincreased (60% of cells had peripheral GM130-labelled puncta inaddition to the central Golgi staining, compared with 30% undercontrol conditions), confirming that the protein undergoes contin-uous and fast recycling through the IC. Qualitatively, however, thesituation was similar to that observed at 3237 C. Also at 15 C,only a subpopulation of the IC elements was filled with VSV-G,including the less peripheral and larger stations (the L-IC), andwere positive for GM130 (Fig. 6A). At the same time, the colocal-ization of the recycling proteins KDEL-R and ERGIC53 withGM130 remained partial at 15 C. KDEL-R and ERGIC53 werepresent in the small, very peripheral spots, as well as in larger, morecentral spots; GM130 was exclusively in the latter (Fig. 6A). The

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Figure 7 p115 and GRASP65 control the dynamics of GM130-positive L-IC

stations and tubules. A, Dynamics of GFPG95 in living COS7 cells (see also

Supplementary information movie 4). Individual images from a movie of a cell

expressing GFPG95, a truncated form of GM130 unable to bind p115. Time is

given in min.sec. Individual spots and tubules are indicated with filled arrowheads.

Note the abundance of peripheral structures containing GFPG95. Frames

00.0800.16 show a tubule originating from a peripheral L-IC, moving towards, and

fusing with another peripheral L-IC. Frames 00.4001.15 show a tubule originating

from the Golgi area, moving retrogradely and becomes a peripheral L-IC station. B,G95 induces the tubular transformation of the IC. COS7 cells were transfected with

FlagG95 and double-stained with anti-Flag antibodies (a, c) and anti-GM130 (b) or

anti-p23 (d) antibodies. Note the tubular staining pattern of GM130 and p23 in cells

expressing FlagG95 as compared with control cells. C, Masking the GM130-bind-

ing site of p115 with GM130/300N (a truncated form of GM130 that can bind

p115, but not GRASP65) redistributes p115 from the Golgi to the E-IC elements.

COS7 cells were transfected with FlagGM130/300N and double stained with anti-

Flag (a) and anti-p115 (b) antibodies. Note that GM130/300N does not localize to

the Golgi or central L-IC but on peripheral puncta (two of which are indicated with

filled arrowheads), there colocalizing with p115. Also, in cells expressing

GM130/300N, p115 is redistributed to the periphery (b), suggesting that the dock-ing/fusion of the E-IC with the more central L-ICs, and/or the Golgi area, is inhibit-

ed. Scale bars,A, 10 m; B, 20 m; C, 12 m.



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only difference was that GM130, which does not colocalize at theperiphery with the ERES marker SEC13 at 37 C, did colocalizewith SEC13 at 15 C, although partially and only at the level of thelarger SEC13-positive units. These findings are consistent with therecent observation that the ERES and the IC elements, which aredistinguishable at 37 C, can mix at 15 C (ref. 22).

When the cells previously incubated at 15 C were warmed (to32 C or 37 C), there was a massive proliferation of the GM130-positive tubules starting from the peripheral puncta. Most tubules

contained VSV-G (Fig. 6A, mo) and anterogradely transportedboth GM130 and VSV-G to the Golgi complex, as indicated by thefast refilling from peripheral puncta of the central Golgi area afterits photobleaching (Fig. 6B; and Supplementary Informationmovie 3). In addition, these GM130 tubules did not colocalize withERGIC53-labelled tubules that also proliferate after removal of the15 C block and, in contrast, were mainly retrograde and directedto the ER (see also refs 10, 26). This distinct behaviour of the tworecycling proteins GM130 and ERGIC53 suggests that they follow

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GFPG95 VSV-G Giantin

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Figure 8. Interfering with GM130-tubule dynamics with G95 inhibits the

transport of VSV-G protein to the Golgi complex. A, COS7 cells were trans-

fected with GFPG95. After overnight expression, the cells were infected with VSV,

incubated at 40 C for 2 h and shifted to 32 C for 15 min (ac), or incubated at

40 C for 2 h, at 15 C for an additional 2 h, and then shifted to 32 C for 5 min

(df). The cells were processed for immunofluorescence and labelled with anti-VSV-

G (b, e) or anti-giantin (c, f) antibodies. Note that 15 min (b) and 5 min (e) after the

shift to 32 C, the VSV-G staining pattern is tubular and/or punctate in G95-trans-

fected cells whereas it is mainly central in control cells, indicating a delay in VSV-G

arrival to the Golgi complex. Note that the general organization of the Golgi com-

plex, as assessed by giantin staining (c, f) is not perturbed in cells expressing G95

at low-to-intermediate levels. Scale bars, 20 m. B, G95 inhibits the transport of

VSV-G from L-IC to the Golgi complex. COS7 cells transfected with GFPG95 (G95)

or with GFP alone (control) were infected with VSV, incubated at 40 C for 2 h and

shifted to 32 C for the indicated times (a), or incubated at 40 C for 2 h, at 15 C

for an additional 2 h, and then shifted to 32 C for the indicated times (b). The cells

were fixed, processed for immunofluorescence and stained with anti-VSV-G anti-

body. At each time point, 200 cells were analysed for the VSV-G staining pattern

and classified as having a punctate pattern when they possessed more than five (in

a) or ten (in b) peripheral structures (tubules or puncta) containing VSV-G. The

results are expressed as percentage of cells exhibiting a punctate pattern of VSV-G

staining and are the means (SD) of three independent experiments. Note that at

time 0 in a very few cells, if any, presented a punctate pattern, whereas in b

almost every cell had a punctate pattern. At late times, few cells had puncta. These

puncta were devoid of GM130, and probably represented post-Golgi transport inter-

mediates. C, The L-IC is a subdomain of the IC, characterized by the presence of IC

markers and the cis-Golgi matrix proteins GM130 and GRASP65, by the absence of

the cis-Golgi markers mannosidase I and HP lectin, and by the propensity to gener-

ate tubules that connect L-IC units to each other and to the Golgi. The transition

from E-IC (containing IC markers but not GM130 and GRASP65) to L-IC (dotted cir-

cles) occurs mainly by fusion of E-IC with GM130-positive tubules (a) or L-IC sta-

tions (b). The incorporation of L-IC into the Golgi complex (rectangles), might

involve one (or both) of two mechanisms: either fusion of L-IC with the cis-Golgi cis-

terna (c), or juxtaposition and flattening of L-IC under the cis-most cisterna (d).



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different recycling pathways and that the GM130 recycling loop isshorter than that of ERGIC53 and operates between the Golgi andL-IC.

Interestingly, the L-IC was more developed and the GM130tubulation was more pronounced (both in terms of number ofGM130-positive tubules per cell and number of cells presentingGM130-positive tubules) in VSV-infected cells compared with con-trol cells (or VSV-infected cells treated with cycloheximide; Table 1and Fig. 4A, e) under temperature conditions that are permissivefor the ER-to-Golgi transport of VSV-G. These results are consis-tent with the possibility that the abundance of the L-IC mightreflect the activity rate of the secretory pathway, and suggest thattubulation of GM130 might be controlled by the presence of cargoinside the IC.Molecular interactions regulating GM130 recycling between theGolgi complex and the L-IC. The main proteins that interact withGM130 are GRASP65 and p115. GRASP65 is tightly associated withthe Golgi membranes, forms a very stable complex with GM130,and is responsible for GM130 association with Golgi membranes6.p115 was originally identified as a protein involved in intra-Golgitransport and transcytosis31,32, associates with GM130 and othermatrix proteins (such as giantin, and golgin160; P.M. andM.A.D.M., unpublished observations), is localized on the Golgi

complex and at the IC, and participates, together with GM130 andGRASP65, in tethering complexes thought to function in mem-brane transport and in cisternae stacking at the Golgi-complexlevel7,8,33,34. We investigated whether the interaction of GM130 withthese proteins controls the dynamics of the GM130-containing L-IC. Because we know that GM130 interacts with GRASP65 andp115 via its carboxy and amino termini, respectively35,36, we con-structed protein mutants that would disrupt these interactions.

First,we studied the distribution of a truncated form of GM130,G95, that lacks the p115-binding domain, but contains theGRASP65-binding domain. GFPG95 localized mainly on periph-eral tubules, but also at the central Golgi (Fig. 7A). The GFPG95tubules were extremely dynamic and moved and extended with aspeed similar to that of GFPGM130 tubules (Fig. 7A andSupplementary Information movie 4). However, when compared

with GM130 tubules, they persisted longer (their average lifespanwas 14 s compared with 5 s for GM130 tubules). The G95 tubulesalso showed more numerous and random changes in directionbefore projecting towards, contacting, and eventually disappearinginto, other puncta or the central Golgi area. In fact, the G95 tubuleshad prevailing lateral direction, and a less obvious anterogradedirection when compared with the GM130 tubules (Fig. 7A; andSupplementary Information movie 4). The above pattern was notexclusive to GFPG95 but was also observed with other G95 con-structs such as FlagG95 (Fig. 7B). One explanation for the effect ofG95 might be the inability of the truncated G95 protein to bindp115, and thus mediate the effective docking of the tubules to theGolgi complex. The fact that the block was not complete could bedue to the inability of G95 to completely inhibit the action of theendogenous nontruncated GM130 protein, or redundant docking

mechanisms.We next studied the distribution of a truncated form of GM130,

GM130/300N, which lacks the GRASP65-binding site but containsthe p115-binding site. GM130/300N did not localize to the centralGolgi or to the tubules, indicating that GRASP65 mediates the asso-ciation of GM130 with Golgi6 and pre-Golgi membranes (Fig. 7C).In fact, probably because of its ability to bind p115, GM130/300Ncolocalized with p115 on peripheral elements of the IC (E-IC), withwhich p115 associates in a GM130-independent way37.Interestingly, GM130/300N caused a redistribution of p115 fromthe Golgi area to the peripheral E-IC elements (Fig. 7C). Theseresults indicate that interfering with the interaction of p115 withendogenous GM130 (the distribution of which at the Golgi and L-IC was unchanged by GM130/300N, data not shown) interfereswith the normal p115 cycling between the Golgi and the periphery.

It has been recently proposed that p115 cycles through pre-Golgicompartments in an exclusively membrane-associated form38 andis not released into a cytosolic pool. Therefore, interfering with theinteraction of p115 with GM130 inhibits the progression of p115-positive E-IC to later and more central stations and suggests thatthe docking of E-IC with L-IC elements (both tubules and stations)might be controlled by p115.

Another truncated form of GM130, G95/GRASP65, which lacksboth the p115- and GRASP65-binding sites, is completely distrib-uted in the cytosol (data not shown), confirming that GRASP65mediates the association of GM130 with the Golgi and pre-Golgimembranes.

Together these results indicate that the interaction betweenGM130 and p115 is required for the anterograde transport anddocking of L-IC elements to the central Golgi and suggest that itmight also control the docking of E-IC to L-IC elements.GM130-positive tubules mediate the incorporation of L-ICs andcargo into the Golgi complex. We next investigated how theGM130p115 interaction functions in ER-to-Golgi transport. Weanalysed the impact of G95, a truncated GM130, on the transportof neosynthesized VSV-G to the Golgi complex (Fig. 8). The secre-tion of VSV-G from the ER and its arrival to the E-IC and L-ICwere not affected, whereas its arrival at the Golgi complex was

inhibited in G95-transfected cells, as compared with control cells(either nontransfected cells or cells transfected withGFPG95/GRASP65 or GFP alone). After 15 min at 32 C in con-trol cells,VSV-G was mainly concentrated in the Golgi; only 25% ofcells presented a consistent fraction of VSV-G in peripheral punc-ta, in contrast to 84 15% of the cells transfected with G95, inwhich VSV-G was mainly present in peripheral puncta and tubuleswith lower Golgi staining (Fig. 8A, ac). This different distributionwas maintained at later time points (45 min) when 55 8% ofG95-transfected cells and 15 3% of control cells presented VSV-G-positive peripheral puncta and tubules, indicating a defect in thetransport to the Golgi system (Fig. 8B, a). Similarly, in cells kept at15 C for 2 h and then shifted to 32 C, the G95-transfected cellshad a slower rate of transport of VSV-G to the Golgi comparedwith control cells (Fig. 8A, df). Under these conditions after 5 min

at 32 C, 86 10% of G95-transfected cells had a large proportionof VSV-G still in the peripheral puncta, whereas in 88 9% of con-trol cells the main fraction of VSV-G was in the central Golgi area(Fig. 8B, b). This difference in VSV-G distribution was maximalafter 5 and 12 min at 32 C and decreased at later time points (30min).

Interfering with the dynamics of GM130 tubules impairs theIC-to-Golgi transport of cargo. G95 tubules have a longer half-lifecompared with GM130 tubules, and are defective in docking andfusing with the Golgi complex. This docking defect might explainwhy the cargo is not efficiently delivered from the L-IC to the cen-tral Golgi complex.

Discussion

The main finding in this report is that the cis-Golgi proteinsGM130 and GRASP65 cycle via membrane tubules through spe-cialized stations of the IC, the L-IC. This L-IC does not contain cis-Golgi markers (such as mannosidase I and HP lectin binding pro-teins) and contains, at any given time, only a relatively minor por-tion (20%) of GM130 and GRASP65, with the rest of the proteinsremaining associated with the central Golgi complex. However, thetwo pools of GM130 are not functionally separated, rather, theyexchange rapidly, as indicated by the fluorescence loss in theperipheral pool induced by bleaching the central pool and viceversa, and by the fast recovery of the fluorescence of the central andperipheral pool after bleaching.

The L-IC represents a subdomain of the IC where the transi-tion between the IC and Golgi occurs. L-IC stations are present inthe central Golgi area as well as in the cell periphery. Although




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physically separated, they are interconnected by membranoustubules establishing functional and often physical continuityamong the various central and peripheral stations. As a result, theL-IC stations probably function as a homogeneous, well-mixedcompartment (Fig. 8C). Morphologically and compositionally, theL-IC stations are similar to traditional IC elements in that theyexhibit the typical IC conformation of convoluted tubular clusters,and contain IC markers. However, they also differ from the E-ICelements, by the presence of the matrix proteins GM130 andGRASP65, by having a more central location, and a different role inthe progression of cargo towards the central Golgi. This is apparentas they receive ER-derived cargo with a delay (of 34 min) from itsappearance in the ERES and E-IC and before its arrival at the cen-tral Golgi complex. The transfer of cargo from E-IC to L-IC occursmainly through a direct fusion of E-IC elements with GM130-pos-itive tubules or a GM130-positive L-IC stations (Fig. 8C, a and b).However, we cannot at present exclude a further possibility, that thetransfer from E-IC to L-IC might occur by a dissociative process inwhich cargo-containing carriers detach from E-IC and are trans-ported to the L-IC. The L-IC is thus analogous to late endosomes,which receive cargo from early endosomes and send it to lysosomesor to the Golgi. Our working model is that cargo must be firstincorporated into the GM130-containing L-IC before entering the

Golgi stack. This entry step is a critical transition in the secretoryprocess. Because the flat, reticulated, cisterna-like structures of theL-IC contain GM130, we believe that its formation represents thenext step in the entry process. This formation might involve one oftwo alternative mechanisms: either the fusion of L-IC stations withthemselves and the cis-Golgi cisterna, followed by flattening of themembranes; or simply juxtaposition and flattening of L-IC stationsunder the cis-most cisterna (Fig. 8C, c and d). It is logical to thinkthat, in their capacity as Golgi matrix proteins, GM130 andGRASP65 might participate in the flattening and the morphogene-sis of this cisstructure. The availability of GM130 and GRASP65 asmarkers of the interface between the IC and the Golgi should facil-itate the dissection of these critical events.

The carriers that operate in the late transport steps from the ICto the Golgi are either the L-IC stations themselves moving en bloc

towards their destinations, or the membranous tubules emanatingfrom these stations (Fig. 8). The abundance and rapid turnover ofthese tubules suggests they have a major role. They reach their des-tinations in several ways: they can dissociate from the original L-ICstation and dock and fuse with the Golgi; they can move en bloctogether with the L-IC station towards the Golgi complex and sub-sequenly fuse with the Golgi membranes; or they can form morestable connections between the L-IC station and the central Golgiin which the cargo appear to move as a bolus39. A recent studydescribes tubular structures emanating from IC elements (afterrelease from a 15 C block) that have been proposed as specific car-riers for soluble cargo; membrane proteins like VSV-G appear inthese tubular structures only when the proteins are heavily overex-pressed40. These observations differ from ours as we do see themembrane protein VSV-G in GM130-positive tubules even at very

low levels of expression and/or infection. It is possible that the sol-uble GFP-containing tubules described previously40 and the VSV-G- and GM130-containing tubules described here represent twoseparate transport systems. We also found that the association ofGM130 with soluble GFP-containing tubules is poor (data notshown), which is consistent with two transport systems.

Other evidence supports the idea of a major function of thetubules in transport. Interfering with the docking of the tubules tothe Golgi complex with the truncated form of GM130, G95, slowsdown the anterograde transport of VSV-G from the L-IC stations tothe Golgi complex. Our results with G95, which indicate that GM130is involved in ER-to-Golgi transport in intact and living cells, are inline with recent data obtained in semi-intact cells using anti-GM130antibodies33,41. The transport block with G95 is partial,however. Thismight be due to the fact that G95 does not completely inhibit the

action of the endogenous, nontruncated, GM130 protein, or to theexistence of redundant docking mechanisms. That tubules increaseboth in terms of number of tubules per cell and of number of cellswith tubules when the demand for anterograde transport increases(for example, after release of the 15 C block, when the cargo thathas accumulated in the IC has to be transported to the Golgi com-plex) is also consistent with the L-IC stations and tubules beinginvolved in membrane transport to the Golgi complex. Remarkably,the extent of GM130 tubulation correlates with the extent of cargotransport, and seems to be controlled by the amount of cargo insidethe IC the tubulation is more evident in VSV-infected cells com-pared with noninfected cells, and it can be partially prevented byprotein synthesis inhibitors. The mechanisms and sensors underly-ing this regulation have not been identified.

What are the relationships that exist between the GM130-posi-tive L-IC tubule system and the molecular machineries known tooperate in ER-to-Golgi transport (involving COPII, COPI, SNAREs,rab1 and p115)? The GM130-positive L-IC stations contain COPI(but not COPII) components, which are patchily distributed alongthe L-IC-associated tubules. As for the SNARE proteins, it is worthnoting that the localization and dynamics of GM130 resemble thosedescribed for membrin42,43. Unlike other SNAREs involved in ER-to-Golgi transport, such as rbet1 and sec22, membrin is enriched in

mature or Golgi-adjacent IC elements,and cycles between these andthe cis-Golgi42,43. Membrin is thus a good candidate for the controlof docking/fusion events involving L-IC stations and tubules.Finally, the recently reported ability of rab1 to interact with GM130(refs 41, 44) raises the intriguing possibility that the dynamics andthe function of GM130 might be controlled by this small GTPase.p115 plays an important role in ER-to-Golgi transport33,37. It isrecruited to COPII vesicles and interacts with selected SNAREs; thisinteraction is controlled by the small GTPase rab1 (ref. 45). By usingthe truncated forms of GM130 lacking the p115- or GRASP65-binding domains, we have shown that p115 controls two distinctsteps of the transport of GM130-positive, L-IC elements thedocking (and fusion) of L-IC elements to the central Golgi andprobably also the docking of p115-containing E-IC elements to theGM130-containing L-IC stations.

We have identified a specialized domain of the IC, the L-IC,where important maturative events of ER-derived membranesoccur, such as the acquisition of structural Golgi components andthe homotypic fusion between L-IC units and their interconnec-tion via membrane tubules. These biochemical and morphologicalchanges may be important in driving the process of incorporationof neosynthesized membranes into Golgi membranes. The defini-tion of the molecular mechanisms involved in this GM130-regulat-ed tubulation process, the understanding of the regulation ofGM130 dynamics, and the identification of its molecular partnerson the tubules and at the L-IC stations remain challenges for futurestudies.

MethodsReagents and antibodiesAll chemical reagents were of analytical grade or higher and purchased from Sigma (Saint Louis, MO)

unless otherwise specified. Cell-culture media were obtained from Gibco (Grand Island, NY). Two dis-

tinct polyclonal anti-G95 antibodies and the polyclonal anti-giantin antibody were prepared as

described previously46. The polyclonal anti-G95 antibody was affinity purified on GSTG95 immobi-

lized on a glutathione-Sepharose 4B column (Amersham Biosciences, Buckinghamshire, UK). Anti-

p115 polyclonal antibodies were generated by immunizing against a fusion protein between glu-

tathione S-transferase (GST) and a C-terminal fragment of p115 (GSTp115-600C),and affinity puri-

fied on p115-600C. The anti-GM130 and anti-KDEL-R monoclonal antibodies were from

Transduction Laboratories (Lexington, KY) and from StressGen Biotechnologies (Victoria,Canada),

respectively. The monoclonal anti-VSV-G clone P5D4 Cy3-conjugated and the anti-Flagbiotinylated

M5 monoclonal antibodies were from Sigma (Saint Louis, MO). The polyclonal anti-mannosidase II

and anti-mannosidase IA was provided by K. W. Moremen (University of Georgia, USA). The poly-

clonal anti-SEC13 and anti-SEC31 antibodies were provided by B. L. Tang (University of Singapore).

The polyclonal anti-p23 antibody was provided by J. Gruenberg (University of Geneva, Switzerland).

The polyclonal anti-COP antibody was provided by J. Lippincott-Schwartz (National Institutes of

Health, Bethesda, MD). The polyclonal anti-GRASP65 antibody was provided by F. Barr (Max Planck

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articles

Institute for Biochemistry, Germany). The monoclonal anti-giantin and the monoclonal anti-ERGIC53

antibodies were provided by H. P. Hauri (University of Basel, Switzerland). The AlexaT M488 goat anti-

mouse and anti-rabbit IgG (H+L) antibodies were from Molecular Probes (Eugene, OR). The anti-rab-

bit and anti-mouse IgG Cy3 conjugated antibodies were from Sigma. The FluoroLinkT MCyT M5-labelled

goat anti-rabbit and anti-mouse IgG (H+L) antibodies were from Amersham Biosciences.

Cell culture and transfectionCOS7 cells were grown in DMEM supplemented with 10% (v/v) FCS, 100 U ml1 penicillin,

100 g ml1 streptomycin and 2 mM L-glutamine. RBL cells were grown in MEM supplemented with

16% (v/v) FCS,100 U ml1 penicillin,100 g ml1 streptomycin and 2 mM L-glutamine. NRK cells

were grown in DMEM supplemented with 5% (v/v) FCS, 100 U ml1 penicillin,100 g ml1 strepto-

mycin,2 mM L-glutamine and nonessential amino acids. COS7 and NRK cells were plated on glass

coverslips the day before transfection. They were transfected with the Trans Fast Transfection Reagent

(Promega, Madison, WI) in accordance with the manufacturers instructions. Alternatively, resuspend-

ed COS7 cells were transfected with the Gene Pulser Electroprotocol (Bio-Rad, Hercules, CA) in accor-

dance with the manufacturers instructions. The cells were treated 16 h after transfection.

DNA constructsCloning of human GM130 cDNA. Poly(A)+ RNA prepared from QGP-1 (human pancreatic carcino-

ma) cells was used to construct a complementary DNA library in APII bacteriophage.A golgin-95

cDNA fragment46 was prepared by reverse transcription polymerase chain reaction (RT-PCR) from

QGP-1 RNA to obtain a probe for screening. The positive clones were subcloned into the pBluescript

SK plasmid vector. The cDNA clone with the longest insert (3.8 kb, named QSY1111) was subcloned

and subjected to nucleotide sequencing. To obtain the full-length cDNA, we constructed another

cDNA library using a poly(A)+ RNA fraction enriched with 4 kb RNAs that can hybridize with a frag-

ment of QSY1111. Screening 1106 independent clones of the new cDNA library yielded 60 positive

clones. The clone with the longest insert (4.3 kb) was subcloned into the pSG5 vector (Novagen,

Madison, WI) and sequenced.

Full-length human GM130 constructs. These constructs were generated by in-frame insertion of the

EcoRIPmaCI cDNA fragment of pSG5/GM130 intoEcoRI/SmaI-digested pEGFP-C2/pEYFP-C2 vec-

tor (Clontech, Palo Alto, CA) or into the EcoRI/EcoRV-digested pcDNAIII1AB vector, which encodes

the Flag epitope.

Assessment of the p115-binding activity of GFPGM130. Because the GFP protein was inserted at the

N terminus of GM130, and this region is involved in p115 binding, we determined whether the recom-

binant protein was able to bind p115 with an affinity comparable to that of the endogenous GM130

protein. To this end, COS7 cell lysates from cells transfected with an empty vector and cells transfected

with GFPGM130 were either immunoprecipitated with anti-p115 antibodies or passed through an

affinity column on which the p115-600CGST fragment, which contains the GM130-binding site, was

immobilized. In brief, cells transfected with an empty vector or with the GFPGM130 construct were

treated with lysis buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1% (v/v) Triton-X 100,1 mM EDTA

and protease inhibitors). Cell lysates were incubated with preimmune or specific antibodies overnight

at 4 C, then with Protein ASepharose for 1 h at 4 C. The immunoprecipitates were washed exten-

sively (7 in lysis buffer) and analysed by SDS PAGE. Alternatively, cell lysates, prepared as described

above, were incubated with GSTp115-600C immobilized on a glutathione Sepharose column, for 1 h

at 4 C, and,after extensive washes in PBS, the retained proteins were eluted with 1 M glycine and

analysed by SDSPAGE. After electroblotting, the nitrocellulose filters were incubated with anti-

GM130 antibodies and then subjected to chemiluminescence detection. The proteins of interest (that

is, the bands corresponding to the endogenous GM130 (relative molecular mass 130,000; Mr 130K),

and the recombinant GFPGM130 protein (Mr 160K) were quantified by densitometry1, and the

coprecipitated or retained proteins were expressed as percentages of the total protein present in the

extract. In the immunoprecipitation experiments, 14 3% of the total endogenous GM130 protein

and 12 4% of the total recombinant GFPGM130 protein (which was 34-fold more abundant than

endogenous GM130) was coimmunoprecipitated with p115. The efficiency of p115 immunoprecipita-

tion in the different samples was comparable as assessed by testing the immunoprecipitates for the

presence of p115.In the p115-600C affinity column experiments, 12 3% of the endogenous GM130and 10 2% of the recombinant GFPGM130 present in the lysates were specifically retained.

Truncated human GM130 constructs. The G95 constructs46, encoding amino acids 371990, were

obtained by subcloning G95 into the pEGFP-C1 vector and into the pcDNAIII1AB vector. The recom-

binant protein was able to bind GRASP65 but not p115, as assessed in immunoprecipitation studies

performed in transfected COS7 cells (see above). The G95/GRASP65 construct, which encodes

residues 371961, was generated by removing the SmaI fragment from the pEGFP-C1/G95 vector. The

GM130/300N constructs, which encode residues 1307, were generated by in-frame insertion of the

HindIII cDNA fragment of pEYFP-C2/GM130 into either the HindIII restriction site of the pEGFP-C2

or the HindIII restriction site of the pcDNAIII 1AB.

CFPVSV-G. The cDNA covering the entire coding region of the temperature-sensitive VSV-G

protein47 was inserted into the EcoRI/SalI-digested pECFP-N1 plasmid (Clontech).

Immunofluorescence analysesCOS7 and NRK cells grown on glass coverslips were fixed in 4% paraformaldehyde in PBS for 10 min

at room temperature and processed for immunofluorescence as described previously2. The HP staining

was performed in accordance with ref. 48. In selected experiments, COS7 cells were treated with

extraction buffer (100 mM MES, pH 6.5, 1 mM EGTA, 0.5 mM MgCl2,1 mM NaN3, and proteaseinhibitors) for 10 min at room temperature. Analysis of VSV-G protein transport was performed in

cells infected with the ts045 VSV strain or transfected with GFPVSV-G. COS7 and NRK cells,plated

on coverslips, were infected with VSV at 32 C for 45min, incubated at 40C for 2 h, and then either

shifted to 32 C for different times or further incubated at 15 C for 2 h (to permit accumulation of the

protein in the IC) before being shifted to 32 C for different times. At the end of the incubations, the

cells were fixed and processed for immunofluorescence.

Quantification of VSV-G transport was performed by analysing the immunostaining patterns of at

least 200 cells in three separate experiments. The results are given in the figure legends or in the text, as

percentage (s.d.) of cells presenting a given pattern.

For confocal imaging, samples were examinated with a ZEISS LSM 510 confocal microscope.

Optical sections were obtained under a 63 oil-immersion objective, at a definition of 1,024 1,024

pixels, adjusting the pinhole diameter to 1 Airy unit for each emission channel.

Time-lapse laser-scanning confocal microscopy (LSCM)GFP-transfected cells, grown in HEPES-buffered medium on glass-bottom Petri dishes, were imaged

with a ZEISS-LSM-510 confocal microscope equipped with a thermoregulation device.

Movies were produced under a 63 oil-immersion objective, at a definition of 512 512 pixels,

with the pinhole diameter set at its maximum value. The GFP was excited with a 488 nm laser line andimaged through a 505550 band-pass filter.For CFP, 458-nm (excitation) and 475525-nm (band

pass) emission filters were used. For YFP, 514-nm (excitation) and 530-nm (long pass) emission filters

were used. The line-average mode was selected,and the number of averages was set to 1. The scan

speed was the maximum, and for double imaging of the same sample the multitrack configuration was

applied. For photobleaching, each round consisted of 100200 iterations (depending on the area to be

bleached) at maximum-intensity laser beam of the relevant wavelength.Movie files were converted to

TIFF format by Graphic Converter 4.0,e dited by NIH Image 1.6.2, and again converted into

QuickTime format.

Quantification of colocalizationConfocal images of double- or triple-labelled COS7 cells were acquired, exported as JPEG files, and

analysed for fluorescent signal overlap. Using Adobe Photoshop 5, a grid (each grid square covered 1%

of the image) was projected onto the image of each channel, and all fluorescent structures were count-

ed. Signals were considered as individual structures if they consisted of spots of intensity values >50

(in a range of 0255). Then the merged images were analysed and the colocalizing structures were

counted. At least 10 cells for sample were analysed, counting from 15 to 250 structures (depending on

the examined marker) per cell.

Correlative light immuno-electron microscopy (CLEM) and immuno-electron

microscopyCOS7 cells were grown on CELLocate coverslips (Eppendorf, Germany) in DMEM supplemented with

10% FCS and 2 mM L-glutamine. Cells were fixed with 0.05% glutaraldehyde plus 4% paraformalde-

hyde in 0.2 M HEPES (pH 7.4) for 15 min, and then with 4% paraformaldehyde in the same buffer for

30 min. After washing, cells were incubated in 0.2% saponin dissolved in blocking solution (PBS sup-

plemented with 1% BSA and 50 mM NH4Cl) for 30 min, then with polyclonal anti-GM130 antibodies

overnight, in blocking solution,washed and treated with the anti-rabbit IgG conjugated with CY3

(Sigma) for 60 min. After washing, samples were analysed under LSCM and the cell of interest under-

went optical sectioning along the Z-axis, when its position within the coordinated grid was deter-

mined. Next, samples were incubated with nanogold-conjugated Fab fragments of anti-rabbit IgG

(NanoProbes, Yaphank,NY) diluted in blocking solution (1:100) for 60 min, extensively washed and

fixed with 1% glutaraldehyde in 0.2 M HEPES (pH 7.3) for 5 min. Gold particles were enhanced by

GoldEnhancer (NanoProbes) in accordance with the manufacturers instructions. Routinely,the time

of enhancement was about 46 min. After washing samples were treated with 1% osmium tetroxide


Table 1 GM130-positive tubule proliferation during IC-to Golgi

transport ofsecretory cargo

Percentage tubulated cells

15 C Exposed to 32 C 40 C Exposed to 32 C

after 15 C after 40 C

Non-infected 11 3 25 3 20 3 22 4cells (3 1) (18 2) (9 1) (14 2)

VSV-infected 14 2 60 5 29 2 59 4

cells (4 1) (48 4) (8 2) (44 3)

VSV-infected 12 1 17 2 22 3 30 3

cells + CHX (4 1) (6 2) (3 1) (4 1)

COS7 cells were infected (or not) with VSV, were incubated for 2 h at 40 C

and then shifted to 32 C for 10 min. Alternatively they were incubated for

2 h at 40 C, kept for additional 2 h at 15 C and then shifted to 32 C for

3 min. Where indicated, VSV-infected cells subjected to the described tem-

perature shifts were treated with 100 g ml1 cycloheximide (+ CHX) at the

beginning of the 40 C incubation period. At the end of each incubation treat-

ment, the cells were fixed, processed for immunofluorescence, and stained

with anti-GM130 (and anti-VSV-G) antibodies, and the number of tubules percell was quantified. At least 200 cells for each treatment were counted and

classified as tubulated (at least one GM130-positive tubule) or highly tubulat-

ed (more than three GM130-positive tubules; these values are shown in

parentheses). Experiments were carried out in triplicate; results are

means s.d.



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plus 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer (pH 7.3) for 2 h on ice (all incubations

before this step were performed at room temperature) in the dark, then dehydrated, embedded in

Epoxy resin, and polymerized for at least 24 h. The CELLocate coverslips were then dissolved with 40%

hydrofluoric acid, and the samples were intensively washed with buffer and then distilled water.

Finally, the cell of interest was sectioned tangetially. Serial sections (of 100 nm) were collected on slot

grids covered with formvar-carbon supporting film and examined at 80 kV in a Zeiss 109 electron

microscope. The images collected by LSCM and EM were aligned with Adobe Photoshop, and the

structure of interest was identified on the basis of its position in space and its labelling with colloidal

gold.

For the immunoEM colocalization of HP lectin and GM130 a double-labelling protocol was per-

formed using diaminobenzidine reaction and silver enhancement.RBL cells were fixed and permeabi-

lized as described above and incubated with the anti-GM130 polyclonal antibody. Next, cells werewashed and treated with nanogold-conjugated Fab fragment of anti-rabbit IgG (NanoProbes). Gold

particles were enhanced by Silver Enhancer (Aurion, Wageningen, The Netherlands) in accordance

with the manufacters instructions. The enhancement reaction was developed for 60 min at 20 C in

the dark. After washing, cells were incubated with HRP-conjugated HP lectin (diluted 1:20 in blocking

solution) overnight.Afterwards, cells were washed, fixed in 1% glutaraldehyde in phosphate buffer (pH

7.4) for 5 min and treated with diaminobenzidine and H2O2 for 10 min at room temperature in accor-

dance with ref. 25. After washing, samples were treated with 0.5% osmium tetroxide plus 0.75% potas-

sium ferrocyanide in 0.1 M cacodylate buffer (pH 7.3), dehydrated, and embedded in Epoxy resin.

Serial sections (70 nm) were collected on copper grids and examined at 80 kV in a Zeiss 109 electron

microscope. CryoimmunoEM labelling was performed in accordance with ref. 49.

RECEIVED 17 APRIL 2001; REVISED 17 JULY 2001; ACCEPTED 7 SEPTEMBER 2001;

PUBLISHED 9 NOVEMBER 2001.

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Documents

The GM130 and GRASP65 Golgi Proteins