Chris Mullins

Lysosomes are membrane-bound cytoplasmic organelles involved in intracellular protein degradation. They contain an assortment of soluble acid-dependent hydrolases and a set of highly glycosylated integral membrane proteins. Most of the properties of lysosomes are shared with a group of cell type-specific compartments referred to as 'lysosome-related organelles', which include melanosomes, lytic granules, MHC class II compartments, platelet-dense granules, basophil granules, azurophil granules, and Drosophila pigment granules. In addition to lysosomal proteins, these organelles contain cell type-specific components that are responsible for their specialized functions. Abnormalities in both lysosomes and lysosome-related organelles have been observed in human genetic diseases such as the Chediak-Higashi and Hermansky-Pudlak syndromes, further demonstrating the close relationship between these organelles. Identification of genes mutated in these human diseases, as well as in mouse and Drosophila: pigmentation mutants, is beginning to shed light on the molecular machinery involved in the biogenesis of lysosomes and lysosome-related organelles.

Formation of intracellular transport intermediates and selection of cargo molecules are mediated by protein coats associated with the cytosolic face of membranes. Here, we describe a novel family of ubiquitous coat proteins termed GGAs, which includes three members in humans and two in yeast. GGAs have a modular structure consisting of a VHS domain, a region of homology termed GAT, a linker segment, and a region with homology to the ear domain of gamma-adaptins. Immunofluorescence microscopy showed colocalization of GGAs with Golgi markers, whereas immunoelectron microscopy of GGA3 revealed its presence on coated vesicles and buds in the area of the TGN. Treatment with brefeldin A or overexpression of dominant-negative ADP ribosylation factor 1 (ARF1) caused dissociation of GGAs from membranes. The GAT region of GGA3 was found to: target a reporter protein to the Golgi complex; induce dissociation from membranes of ARF-regulated coats such as AP-1, AP-3, AP-4, and COPI upon overexpression; and interact with activated ARF1. Disruption of both GGA genes in yeast resulted in impaired trafficking of carboxypeptidase Y to the vacuole. These observations suggest that GGAs are components of ARF-regulated coats that mediate protein trafficking at the TGN.

Germination provides many potentially unrecognized sources of variation in the regeneration niche. In this study we relate germination requirements and seed size for 16 species of pioneer trees to microclimatic conditions present in gaps in semi-deciduous rain forest in Panama. We found that, whereas increased duration of direct irradiance can be an effective indicator of the presence of a canopy gap across all scales of canopy openness, diel fluctuations in soil temperature effectively discriminate both understory sites and small gaps (25 m2) from larger gaps. Germination response was significantly related to seed size. Small-seeded species (seed mass <2 mg) showed significantly greater germination in response to irradiance of 22.3 μmol·m−2·s−1 than in complete darkness. Their germination was unaffected by an increasing magnitude of diel temperature fluctuation up to a species-specific threshold, above which it declined. Large-seeded species (seed mass >2 mg) germinated equally in light and darkness (with one exception) and either showed a positive germination response to an increasing magnitude of temperature fluctuation (four species) or no significant response (four species). The maximum seed burial depth from which seedlings could emerge successfully was strongly positively associated with seed mass. We conclude that photoblastic germination of tropical pioneer trees results in small-seeded species germinating in gaps only when seeds are located in microsites that are suitable for seedling emergence. A positive germination response to increasing temperature fluctuation can stimulate germination of larger-seeded species in larger gaps and when they are buried beneath an opaque soil or litter layer. Therefore, seed size differences constrain the physiological mechanisms of canopy gap detection in tropical pioneer trees and might contribute to observed differences in the distribution of adult plants in relation to canopy gap size.

Here we report the identification and characterization of AP-4, a novel protein complex related to the heterotetrameric AP-1, AP-2, and AP-3 adaptors that mediate protein sorting in the endocytic and late secretory pathways. The key to the identification of this complex was the cloning and sequencing of two widely expressed, mammalian cDNAs encoding new homologs of the adaptor beta and sigma subunits named beta4 and sigma4, respectively. An antibody to beta4 recognized in human cells an approximately 83-kDa polypeptide that exists in both soluble and membrane-associated forms. Gel filtration, sedimentation velocity, and immunoprecipitation experiments revealed that beta4 is a component of a multisubunit complex (AP-4) that also contains the sigma4 polypeptide and two additional adaptor subunit homologs named mu4 (mu-ARP2) and epsilon. Immunofluorescence analyses showed that AP-4 is associated with the trans-Golgi network or an adjacent structure and that this association is sensitive to the drug brefeldin A. We propose that, like the related AP-1, AP-2, and AP-3 complexes, AP-4 plays a role in signal-mediated trafficking of integral membrane proteins in mammalian cells.