articles NATURE CELL BIOLOGY VOL 3 DECEMBER 2001 http://cellbio.nature.com 1101 The GM130 and GRASP65 Golgi proteins cycle through and define a subdomain of the intermediate compartment Pierfrancesco 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: demattei@cmns.mnegri.it Integrating the pleomorphic membranes of the intermediate compartment (IC) into the array of Golgi cisternae is a crucial step in membrane transport, but it is poorly understood. To gain insight into this step, we investigated the dynamics 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 and a constellation of mobile structures that we call late IC stations. These stations are intermediate between the IC and the cis-Golgi in terms of composition, and they receive cargo from earlier IC elements and deliver it to the Golgi complex. Late IC elements are transient in nature and sensitive to fixatives; they are seen in only a fraction of fixed cells, whereas they are always visible in living cells. Finally, late IC stations undergo homotypic fusion and establish tubular connections between themselves and the Golgi. Overall, these features indicate that late IC stations mediate the transition between IC elements and the cis-Golgi face. T he Golgi apparatus has a complex architecture composed of stacks of flat cisternae connected in a ribbon-like fashion by tubular reticular areas. How such an organization is achieved at the cis-Golgi face and maintained through the Golgi in spite of the continuous flux of membranes remains a central problem in the biology of transport. Two classes of protein complexes are thought to be involved in morphogenesis of the Golgi complex, possibly through a coordinated action: the Golgi spectrin–actin skeleton and the Golgi matrix proteins. The spectrin skeleton assembles on Golgi membranes in a fashion dependent on the activity of the small GTPase ARF (ADP-ribosylation factor) and on the level of phosphatidylinositol 4,5-bisphosphate 1,2 . The spectrin skeleton facilitates incorporation of endoplasmic reticulum (ER)- derived membranes into the cis-Golgi 1 ; it may also control the structure of Golgi stacks, on the basis of the probable similarity of the known role of spectrin at the plasma membrane 3 . The ‘Golgi matrix’ proteins 4,5 were originally identified as a set of insoluble Golgi proteins, and include GM130 and GRASP65. These proteins are involved in postmitotic reassembly and stacking of the Golgi cisternae 6–8 and can assemble in a structure reminiscent of the Golgi even in the absence of Golgi resident proteins suggesting that they can act as primary scaffold components underpinning the Golgi architecture 9 . Remarkably, GM130, GRASP65 and other Golgi matrix proteins are located primarily at the cis-pole of the Golgi stacks 4,6 , where they probably function in the incorporation of the ER-derived membranes into the Golgi. The membranes that reach the cis-Golgi are tubular clusters that constitute the ER–Golgi intermediate compartment (IC) 10 . The biogenesis, maintainance, and identity of the IC have been and are objects of intense study. Current theories suggest that the IC may be considered either as an outgrowth of the ER 11,12 , as a part of the cis- Golgi network (CGN) 13 , or as a separate compartment 14–17 . In the lat- ter case, the IC might be viewed as a stable compartment 15,16 or as a transient compartment made up of transport intermediates operat- ing between the ER and the Golgi 14,17 . The difficulties in reconciling these apparently contrasting views and in defining the boundary of the IC result largely from the disperse distribution and pleomor- phic organization of this compartment, and from its dynamic behaviour 10 . Even the molecular composition of the IC elements is highly heterogenous 18 and includes proteins continuously cycling between the ER and the Golgi complex. However, by combining the broadest definition of the ER–Golgi IC (consisting of the mem- branes interposed between the ER and the Golgi stacks) with the information derived from the dynamics of proteins traversing this compartment (which includes cargo proteins 19,20 , recycling pro- teins 21 , and the coat complexes COPI and COPII 22–24 ), it is possible to distinguish three layers in the IC. The first layer includes the ER exit sites (ERES, or transitional ER), marked by COPII, and made up of rather stationary elements 22,24 . The second layer consists of tubular clusters that move long distances, travelling in centripetal direction along microtubules; this layer contains COPI, but not COPII, and forms what we define from here onwards as IC. The third 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 proteins 25 ). According to the view of the IC as a transient compartment, these three layers correspond to three stages of the IC membrane lifespan: origin at the ERES via a sorting process mediated by the SAR1p–COPII complex; motor- driven translocation towards the Golgi along microtubules and concomitant loss of ER-recycled components; and final incorpora- tion into the Golgi complex. Although the first two stages have been extensively studied and their molecular machineries satisfactorily elucidated, 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 investigated how Golgi-structural components such as the matrix proteins GM130 and GRASP65 associate with the incoming IC membranes. We find that IC elements acquire these Golgi components before contacting the cis-pole of the central Golgi area, at the level of a © 2001 Macmillan Magazines Ltd