Biogenesis of tubular ER-to-Golgi transport intermediates.

• 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 approximately 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 GTPgammaS 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.

Nyckelord

Animals

Cercopithecus aethiops

Endoplasmic Reticulum

metabolism

Golgi Apparatus

metabolism

Green Fluorescent Proteins

analysis

Hela Cells

Humans

Mannose-Binding Lectins

metabolism

Membrane Proteins

metabolism

Microtubules

physiology

ultrastructure

Models

Biological

Receptors

Peptide

metabolism

Recombinant Fusion Proteins

analysis

Transport Vesicles

physiology

ultrastructure

Vero Cells

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)