Abstract: The trans Golgi network (TGN) and endosomes are key sites of protein transport within cells. Proteins with various destinies such as secretion, degradation in the lysosome (yeast vacuole), or residency at different organelles pass through the TGN/endosomes. Clathrin plays a fundamental role in ferrying proteins between these organelles, assisted by adaptors that select cargo for transport, link clathrin to membranes, and recruit accessory factors that provide additional functions. Two major adaptors that facilitate transport at the TGN/endosomes are the AP-1 complex and the Gga family of proteins. Here I describe a conserved AP-1 accessory factor of the yeast Saccharomyces cerevisiae (Laa1p, for large AP-1 accessory) and also explore the role of ubiquitin in sorting proteins at endosomes. Laa1p functions in clathrin-mediated transport specifically in conjunction with AP-1. A deletion of the LAA1 gene exacerbated growth and transport defects of cells that carry either a temperature-sensitive allele of clathrin heavy chain or deletions of GGA genes. Laa1p preferentially associated with AP-1 over Gga2p in protein interaction and colocalization experiments. Furthermore, AP-1 visualized by fluorescence microscopy was mislocalized in cells lacking LAA1. The predicted structure of Laa1p is homologous to the core of AP-1, raising the possibility that Laa1p, like AP-1, interacts with lipids and/or the small membrane-localized GTPase Arf. Consistent with the latter, Laa1p localization was disrupted by inactivation of Arf. Thus, the evidence suggests that Laa1p may stabilize AP-1 localization by bridging an interaction between the adaptor and membrane-bound Arf. Proteins are commonly directed to the vacuole/lysosome from the TGN and endosomes by virtue of an attached ubiquitin moiety, but mounting evidence indicates that ubiquitin in trans to cargo is also important for sorting. Here I demonstrate that cis ubiquitin-independent cargo is missorted in ubiquitin-depleted cells that also lack the deubiquitinating enzyme Doa4p; ubiquitin depletion by deleting structural ubiquitin genes did not cause missorting. Cells lacking cofactors of the ubiquitin ligase Rsp5p (Bul1p and Bul2p) also missorted cis ubiquitin-independent cargo, albeit moderately. The results are consistent with a model where sorting is regulated by cycles of ubiquitination of machinery by Rsp5p followed by Doa4p-dependent deubiquitination.