Three distinct adaptor protein (AP) complexes involved in protein trafficking have been identified. AP-1 and AP-2 mediate protein sorting at the trans-Golgi network and plasma membrane, respectively, whereas the function of AP-3 has not been defined. A screen for factors specifically involved in transport of alkaline phosphatase (ALP) from the Golgi to the vacuole/lysosome has identified Ap16p and Ap15p of the yeast AP-3 complex. Deletion of each of the four AP-3 subunits results in selective mislocalization of ALP and the vacuolar t-SNARE, Vam3p (but not CPS and CPY), while deletion of AP-1 and AP-2 subunits has no effect on vacuolar protein delivery. This study, therefore, provides evidence that the AP-3 complex functions in cargo-selective protein transport from the Golgi to the vacuole/lysosome.

Functional polymorphism in genes can be classified as coding variation, altering the amino-acid sequence of the encoded protein, or regulatory variation, affecting the level or pattern of expression of the gene. Coding variation can be recognized directly from DNA sequence, and consequently its frequency and characteristics have been extensively described. By contrast, virtually nothing is known about the extent to which gene regulation varies in populations. Yet it is likely that regulatory variants are important in modulating gene function: alterations in gene regulation have been proposed to influence disease susceptibility and to have been the primary substrate for the evolution of species. Here, we report a systematic study to assess the extent of cis-acting regulatory variation in 69 genes across four inbred mouse strains. We find that at least four of these genes show allelic differences in expression level of 1.5-fold or greater, and that some of these differences are tissue specific. The results show that the impact of regulatory variants can be detected at a significant frequency in a genomic survey and suggest that such variation may have important consequences for organismal phenotype and evolution. The results indicate that larger-scale surveys in both mouse and human could identify a substantial number of genes with common regulatory variation.

an intermediate compartment en route to the lysosome (Kornfeld and Mellman, 1989;Kornfeld, 1992). In More than 40 vacuolar protein sorting (vps) mutantsSaccharomyces cerevisiae, a similar receptor-mediated have been identified which secrete proenzyme forms machinery exists for sorting of the soluble hydrolase of soluble vacuolar hydrolases to the cell surface. A carboxypeptidase Y (CPY) to the lysosome/vacuole. CPY subset of these mutants has been found to show selective transits the early secretory pathway in precursor form. defects in the sorting of two vacuolar membrane Upon arrival in the late Golgi, a sorting signal in precursor proteins. Under non-permissive conditions, vps45 tsf CPY is recognized by a transmembrane receptor, Vps10p, (SEC1 homolog) and pep12/vps6 tsf (endosomal resulting in diversion of CPY from bulk protein flow to t-SNARE) mutants efficiently sort alkaline phosphatase the cell surface via packaging of precursor CPY into (ALP) to the vacuole while multiple soluble vacuolar vesicles bound for the endosome (Marcusson et al., 1994). proteins and the membrane protein carboxypeptidaseMutations in the vacuolar sorting signal of precursor yscS (CPS) are no longer delivered to the vacuole.CPY result in secretion of mutant CPY to the cell surface Vacuolar localization of ALP in these mutants does (Johnson et al., 1987;Valls et al., 1987), and several not require transport to the plasma membrane followed genetic approaches have been employed to detect vacuolar by endocytic uptake, as double mutants of pep12 tsf and protein sorting (vps) mutants which missort and secrete vps45 tsf with sec1 and end3 sort and mature ALP at the soluble vacuolar hydrolases (Bankaitis et al., 1986; non-permissive temperature. Given the demonstrated Robinson et al., 1988). To date, Ͼ40 vps mutant complerole of t-SNAREs such as Pep12p in transport vesicle mentation groups have been identified; in each of these recognition, our results indicate that ALP and CPS mutants, delivery of CPY to the vacuole is compromised. are packaged into distinct transport intermediates.The majority of vps mutants also mislocalize the soluble Consistent with ALP following an alternative route to hydrolases proteinase A (PrA) and proteinase B (PrB). the vacuole, isolation of a vps41 tsf mutant revealed that Not all vacuolar proteins are affected by mutations in at non-permissive temperature ALP is mislocalized VPS genes. For example, the type II integral vacuolar while vacuolar delivery of CPS and CPY is maintained. membrane protein-repressible alkaline phosphatase (ALP)

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Transport of proteins through the ALP (alkaline phosphatase) pathway to the vacuole requires the function of the AP-3 adaptor complex and Vps41p. However, unlike other adaptor protein-dependent pathways, the ALP pathway has not been shown to require additional accessory proteins or coat proteins, such as membrane recruitment factors or clathrin. Two independent genetic approaches have been used to identify new mutants that affect transport through the ALP pathway. These screens yielded new mutants in both VPS41 and the four AP-3 subunit genes. Two new VPS41 alleles exhibited phenotypes distinct from null mutants of VPS41, which are defective in vacuolar morphology and protein transport through both the ALP and CPY sorting pathways. The new alleles displayed severe ALP sorting defects, normal vacuolar morphology, and defects in ALP vesicle formation at the Golgi complex. Sequencing analysis of these VPS41 alleles revealed mutations encoding amino acid changes in two distinct domains of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain repeat (CHCR) domain. We demonstrate that the N-terminus of Vps41p is required for binding to AP-3, whereas the C-terminal CHCR domain directs homo-oligomerization of Vps41p. These data indicate that a homo-oligomeric form of Vps41p is required for the formation of ALP containing vesicles at the Golgi complex via interactions with AP-3.

Genetic analyses of vacuolar protein sorting in Saccharomyces cerevisiae have uncovered a large number of mutants (vps) that missort and secrete soluble vacuolar hydrolases. Here we report the characterization of the gene product affected in one of these mutants, Vps8p. Polyclonal antiserum raised against a trpE-Vps8 fusion protein specifically detects a 134-kDa protein in labeled yeast cell extracts. Subcellular fractionation studies demonstrate that Vps8p is distributed between a low speed membrane pellet fraction and a high speed membrane pellet fraction. The lack of a hydrophobic domain in Vps8p suggests that Vps8p peripherally associates with a membrane(s). This association was found to depend on the function of Vps21p, a member of the Rab/ Ypt/Sec4 family of small GTPases. In vps21 null mutant cells, Vps8p is found in the cytosol. In addition, overexpression of Vps21p partially suppresses a vps8 null mutant, indicating that Vps8p and Vps21p functionally interact. Vps8p contains a C-terminal cysteine-rich region that conforms to the H2 variant of the RING finger Zn 2؉ binding motif. Truncation of this C-terminal region partially compromises Vps8p function. While vps8 null mutant strains missort and secrete soluble vacuolar hydrolases, the integral vacuolar membrane protein, alkaline phosphatase (ALP), is sorted to the vacuole and matured normally. In addition, when vps8 mutants are combined with endocytic or late secretory pathway mutants (end3 or sec1, respectively), ALP is still delivered to the vacuole. These observations indicate that ALP is sorted to the vacuole in a Vps8p-independent manner, possibly via an alternative vesicle carrier.

Genetic analyses of vacuolar protein sorting in Saccharomyces cerevisiae have uncovered a large number of mutants (vps) that missort and secrete vacuolar hydrolases. A small subset of vps mutants exhibit a temperature-conditional growth phenotype and show a severe defect in the localization of soluble vacuolar proteins, yet maintain a near-normal vacuole structure. Here, we report on the cloning and characterization of the gene affected in one of these mutants, VPS45, which has been found to encode a member of a protein family that includes the yeast proteins Sec1p, Sly1p and Vps33p, as well as n-Sec1, UNC18 and Rop from other eukaryotic organisms. These proteins are thought to participate in vesicle-mediated protein transport events. Polyclonal antiserum raised against a TrpE-Vps45 fusion protein specifically detects a stable 67 kDa protein in labeled yeast cell extracts. Subcellular fractionation studies demonstrate that the majority of Vps45p is associated with a high-speed membrane pellet fraction that includes Golgi, transport vesicles and, potentially, endosomal membranes. Significantly, this fraction lacks ER, vacuole and plasma membranes. Overexpression of Vps45p saturates the sites with which Vps45p associates. A vps45 null mutant accumulates vesicles, many of which were found to be present in large clusters. This accumulation of potential transport vesicles indicates that Vps45p may facilitate the targeting and/or fusion of these vesicles in the vacuolar protein sorting pathway.