Deletion of the clathrin heavy-chain gene,

Metalloprotease disintegrins (a disintegrin and metalloprotease (ADAM) and metalloprotease, disintegrin, cysteine-rich proteins (MDC)) are a family of membraneanchored glycoproteins that function in diverse biological processes, including fertilization, neurogenesis, myogenesis, and ectodomain processing of cytokines and other proteins. The cytoplasmic domains of ADAMs often include putative signaling motifs, such as prolinerich SH3 ligand domains, suggesting that interactions with cytoplasmic proteins may affect metalloprotease disintegrin function. Here we report that two SH3 domain-containing proteins, endophilin I (SH3GL2, SH3p4) and a novel SH3 domain-and phox homology (PX) domain-containing protein, termed SH3PX1, can interact with the cytoplasmic domains of the metalloprotease disintegrins MDC9 and MDC15. These interactions were initially identified in a yeast two-hybrid screen and then confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells expressing both binding partners. SH3PX1 and endophilin I both preferentially bind the precursor but not the processed form of MDC9 and MDC15 in COS-7 cells. Since rat endophilin I is thought to play a role in synaptic vesicle endocytosis and SH3PX1 has sequence similarity to sorting nexins in yeast, we propose that endophilin I and SH3PX1 may have a role in regulating the function of MDC9 and MDC15 by influencing their intracellular processing, transport, or final subcellular localization.Metalloprotease disintegrins (also referred to as ADAMs, 1 a disintegrin and metalloprotease, or MDC proteins, metalloprotease, disintegrin, cysteine-rich proteins) are a family of glycoproteins related to snake venom metalloproteases and integrin ligands (1-3). They are composed of several domains including a pro-domain, a metalloprotease domain, a disintegrin domain, a cysteine-rich region, and in most cases a membrane-spanning region and cytoplasmic tail. Metalloprotease disintegrins have been shown to play a role in fertilization (4 -8), muscle cell binding and fusion (9), shedding of tumor necrosis factor ␣, and other membrane-anchored proteins from the plasma membrane (10 -14), regulating neurogenesis and the function of Notch (15-18) and Delta (19), and processing of heparin-binding epidermal growth factor-like growth factor (HB-EGF) (20). Currently 28 ADAMs have been identified, 15 of which contain a catalytic zinc-binding consensus sequence (HEXXH) (3, 21). Four of these ADAMs have been shown to be catalytically active metalloproteases (MADM/Kuz/ADAM 10 (22); tumor necrosis factor ␣-converting enzyme (TACE)/ADAM 17 (10, 11, 23); meltrin-␣/ADAM 12 (24); and MDC9/ADAM 9/meltrin-␥ (20, 25)). Besides their roles as metalloproteases, ADAMs are also thought to mediate cell-cell interactions. This was first suggested by the similarity of both the ␣ and ␤ subunit of the heterodimeric sperm protein fertilin (ADAM1/ADAM2) to snake venom disintegrins (6), which are short soluble peptides that bind to integrins (␣ IIb ␤ 3 and ␣ v ␤ 3 ) and are potent...

Clathrin-mediated vesicular transport is important for normal growth of the yeast Saccharomyces cerevisiae. Previously, we identified a genetic locus (SCDI) that influences the ability of clathrin heavy-chain-deficient (Chc-) which encoded CHCJ, were recovered. One of the suppressor loci was shown to be UBI4, the polyubiquitin gene. UBI4 rescues only in high copy number and is not allelic to SCD1. The conjugation of ubiquitin to intracellular proteins can mediate their selective degradation. Since UBI4 is required for survival of yeast cells under stress and is induced during starvation, ubiquitin expression in GAL1:CHC1 cells was examined. After a shift to growth on glucose to repress synthesis of clathrin heavy chains, UBI4 mRNA levels were elevated >10-fold, whereas the quantity of free ubiquitin declined severalfold relative to that of Chc+ cells. In addition, novel higher-molecular-weight ubiquitin conjugates appeared in clathrin-deficient cells. We suggest that higher levels of ubiquitin are required for turnover of mislocalized or improperly processed proteins that accumulate in the absence of clathrin and that ubiquitin may play a general role in turnover of proteins in the secretory or endocytic pathway.

Clathrin and its associated proteins constitute a major class of coat proteins involved in vesicle budding during membrane transport. An interesting characteristic of the yeast clathrin heavy chain gene (CHC1) is that in some strains a CHC1 deletion is lethal, while in others it is not. Recently, our laboratory developed a screen that identified five multicopy suppressors that can rescue lethal strains of clathrin heavy chain-deficient yeast (Chc - scd1-i) to viability. One of these suppressors, SCD5, encodes a novel protein of 872 amino acids containing two regions of repeated motifs of unknown function. Deletion of SCD5 has shown that it is essential for cell growth at 30 degrees C. scd5-delta strains carrying low copy plasmids encoding C-terminal truncations of Scd5p are temperature sensitive for growth at 37 degrees C. At the nonpermissive temperature, cells expressing a 338-amino acid deletion (Scd5P-delta 338) accumulate an internal pool of fully glycosylated invertase and mature alpha-factor, while processing and sorting of the vacuolar hydrolase carboxypeptidase Y is normal. The truncation mutant also accumulates 80- to 100-nm vesicles similar to many late sec mutants. Moreover, at 34 degrees C, overexpression of Scd5p suppresses the temperature sensitivity of a sec2 mutant, which is blocked at a post-Golgi step of the secretory pathway. Biochemical analyses indicate that approximately 50% of Scd5p sediments with a 100,000 x g membrane fraction and is associated as a peripheral membrane protein. Overall, these results indicate that Scd5p is involved in vesicular transport at a late stage of the secretory pathway. Furthermore, this suggests that the lethality of clathrin-deficient yeast can be rescued by modulation of vesicular transport at this late secretory step.