Do cargo proteins influence this process? Recent work * Department of Molecular and Cell Biology and on the exchange of proteins between the endoplasmic † Howard Hughes Medical Institute reticulum (ER) and the Golgi apparatus in Saccharo-University of California myces cerevisiae and higher eukaryotes suggests that Berkeley, California 94720 all three requirements for functional vesicle budding could be regulated in a single mechanistic step, the formation of a "priming complex" of a small GTPase, a Transport vesicles carry lumenal and membrane cargo membrane protein, and a coat subunit (Figure 1). proteins between secretory organelles in eukaryotic To Begin: Recruit a GTPase cells. To make such a vesicle, cytosolic coat proteins to the Donor Membrane assemble on a donor membrane and deform it into a COPII (coat protein complex II)-coated vesicles transbud. Cargo proteins are sorted into the originating vesiport proteins from the ER to the Golgi. The COPII coat cle, which then separates from the donor membrane, consists of the small GTPase Sar1p and the heteroditravels a certain distance, and finally fuses with the acmeric protein complexes Sec23/24p and Sec13/31p ceptor organelle. (Barlowe, 1998). These five proteins are necessary and At the time of vesicle budding, three specific requiresufficient to produce COPII vesicles from ER microments have to be fulfilled in order to produce a functional somes (Barlowe et al., 1994) and from chemically defined vesicle. First, different vesicular transport processes are liposomes (Matsuoka et al., 1998). mediated by different coat protein complexes, and Proteins can also be recycled to the ER from the cistherefore a donor membrane must attract the correct Golgi via a backward, or retrograde, route. Such proteins species of cytosolic coat proteins. Second, a transport include ER residents that have escaped ER retention, vesicle needs to be equipped with certain membrane and functional components of COPII ("anterograde") proteins that have essential tasks at a later stage. vesicles that return to participate in another round of v-SNAREs, for example, are required for fusion with the COPII vesicle formation. Retrograde vesicles are coated acceptor membrane (reviewed by Nichols and Pelham, with the COPI coat (Cosson and Letourneur, 1997; 1998). These proteins must be included into the vesicles Gaynor et al., 1998), which consists of the small GTPase with high fidelity. And third, cargo proteins have to be recognized and included into the originating vesicles.

We describe recent developments with multifunctional nanoengineered polymer capsules. In addition to their obvious use as a delivery system, multifunctional nanocontainers find wide application in enzymatic catalysis, controlled release, and directed drug delivery in medicine. The multifunctionality is provided by the following components: 1) Luminescent semiconductor nanocrystals (quantum dots) that facilitate imaging and identification of different capsules, 2) superparamagnetic nanoparticles that allow manipulation of the capsules in a magnetic field, 3) surface coatings, which target the capsules to desired cells, 4) metallic nanoparticles in the capsule wall that act as an absorbing antenna for electromagnetic fields and provide heat for controlled release, and 5) enzymes and pharmaceutical agents that allow specific reactions. The unique advantage of multifunctional microcapsules in comparison to other systems is that they can be simultaneously loaded/functionalized with the above components, allowing for the combination of their properties in a single object.

Protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus involves specific uptake into coat protein complex II (COPII)-coated vesicles of secretory and of vesicle targeting (v-SNARE) proteins. Here, two ER to Golgi v-SNAREs, Bet1p and Bos1p, were shown to interact specifically with Sar1p, Sec23p, and Sec24p, components of the COPII coat, in a guanine nucleotide-dependent fashion. Other v-SNAREs, Sec22p and Ykt6p, might interact more weakly with the COPII coat or interact indirectly by binding to Bet1p or Bos1p. The data suggest that transmembrane proteins can be taken up into COPII vesicles by direct interactions with the coat proteins and may play a structural role in the assembly of the COPII coat complex.

Major histocompatibility complex class I proteins play a key role in the recognition and presentation of peptide antigens to the host immune system. The structure of various major histocompatibility complex class I proteins has been determined experimentally in complex with several antigenic peptides. However, the structure in the unbound (empty) form is not known. To study the conformational dynamics of the empty major histocompatibility complex class I molecule comparative molecular dynamics simulations have been performed starting from the crystal structure of a peptide bound class I peptide-binding domain in the presence and absence of a peptide ligand. Simulations including the bound peptide stayed close to the experimental start structure at both simulation temperatures (300 and 355 K) during the entire simulation of 26 ns. Several independent simulations in the absence of peptide indicate that the empty domain may not adopt a single defined conformation but is conformationally significantly more heterogeneous in particular within the alpha-helices that flank the peptide binding cleft. The calculated conformational dynamics along the protein chain correlate well with available spectroscopic data and with the observed site-specific sensitivity of the empty class I protein to proteolytic digestion. During the simulations at 300 K the binding region for the peptide N-terminus stayed close to the conformation in the bound state, whereas the anchor region for the C-terminus showed significantly larger conformational fluctuations. This included a segment at the beginning of the second alpha-helix in the domain that is likely to be involved in the interaction with the chaperone protein tapasin during the peptide-loading process. The simulation studies further indicate that peptide binding at the C- and N-terminus may follow different mechanisms that involve different degrees of induced conformational changes in the peptide-binding domain. In particular binding of the peptide C-terminus may require conformational stabilization by chaperone proteins during peptide loading.

To investigate the factors involved in the sorting of cargo proteins into COPII endoplasmic reticulum (ER) to Golgi apparatus transport vesicles, we have created a strain of S. cerevisiae (p24⌬8) that lacks all eight members of the p24 family of transmembrane proteins (Emp24p, Erv25p, and Erp1p to Erp6p). The p24 proteins have been implicated in COPI and COPII vesicle formation, cargo protein sorting, and regulation of vesicular transport in eukaryotic cells. We find that p24⌬8 cells grow identically to wild type and show delays of invertase and Gas1p ER-to-Golgi transport identical to those seen in a single ⌬emp24 deletion strain. Thus, p24 proteins do not have an essential function in the secretory pathway. Instead, they may serve as quality control factors to restrict the entry of proteins into COPII vesicles.