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Vol. 17, Issue 2, 723-737, February 2006
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* Cell Biology and Biophysics Programme, European Molecular Biology Laboratory, 69117 Heidelberg, Germany;
Department of Medical Biochemistry, Göteborg University, 413 90 Gothenburg, Sweden
Submitted June 29, 2005;
Revised November 8, 2005;
Accepted November 16, 2005
Monitoring Editor: Benjamin Glick
Tubular transport intermediates (TTIs) have been described as one class of transport carriers in endoplasmic reticulum (ER)-to-Golgi transport. In contrast to vesicle budding and fusion, little is known about the molecular regulation of TTI synthesis, transport and fusion with target membranes. Here we have used in vivo imaging of various kinds of GFP-tagged proteins to start to address these questions. We demonstrate that under steady-state conditions TTIs represent 
20% of all moving transport carriers. They increase in number and length when more transport cargo becomes available at the donor membrane, which we induced by either temperature-related transport blocks or increased expression of the respective GFP-tagged transport markers. The formation and motility of TTIs is strongly dependent on the presence of intact microtubules. Microinjection of GTP
S increases the frequency of TTI synthesis and the length of these carriers. When Rab proteins are removed from membranes by microinjection of recombinant Rab-GDI, the synthesis of TTIs is completely blocked. Microinjection of the cytoplasmic tails of the p23 and p24 membrane proteins also abolishes formation of p24-containing TTIs. Our data suggest that TTIs are ER-to-Golgi transport intermediates that form preferentially when transport-competent cargo exists in excess at the donor membrane. We propose a model where the interaction of the cytoplasmic tails of membrane proteins with microtubules are key determinants for TTI synthesis and may also serve as a so far unappreciated model for aspects of transport carrier formation.
Abbreviations used: BFA, brefeldin A; COP, coat protein; ER, endoplasmic reticulum; GDI, GTPase dissociation inhibitor; GFP, green fluorescent protein; IC, intermediate compartment; KDEL-R, KDEL receptor; TGN, trans-Golgi network; TTIs, tubular transport intermediates; VSV-G, vesicular stomatitis viral glycoprotein.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
Address correspondence to: Jeremy C. Simpson (simpson{at}embl.de) or Rainer Pepperkok (pepperko{at}embl.de).
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