Characterization of Mammalian Ecm29, a 26 S Proteasome-associated Protein That Localizes to the Nucleus and Membrane Vesicles

Abstract

In addition to its thirty or so core subunits, a number of accessory proteins associate with the 26 S proteasome presumably to assist in substrate degradation or to localize the enzyme within cells. Among these proteins is ecm29p, a 200-kDa yeast protein that contains numerous HEAT repeats as well as a putative VHS domain. Higher eukaryotes possess a well conserved homolog of yeast ecm29p, and we produced antibodies to three peptides in the human Ecm29 sequence. The antibodies show that Ecm29 is present exclusively on 26 S proteasomes in HeLa cells and that Ecm29 levels vary markedly among mouse organs. Confocal immunofluorescence microscopy localizes Ecm29 to the centrosome and a subset of secretory compartments including endosomes, the ER and the ERGIC. Ecm29 is up-regulated 2-3-fold in toxinresistant mutant CHO cells exhibiting increased rates of ER-associated degradation. Based on these results we propose that Ecm29 serves to couple the 26 S proteasome to secretory compartments engaged in quality control and to other sites of enhanced proteolysis.

A Protein Interaction Network for Ecm29 Links the 26 S Proteasome to Molecular Motors and Endosomal Components

“…Consistent with a possible adaptor function for Ecm29, Ecm proteasomes are localized on the endoplasmic reticulum (ER), on endosomes and at the centrosome in HeLa cells; based on its intracellular distribution we proposed that Ecm29 links 26 S proteasomes to these cellular compartments (30). Here we report that genome-wide two-hybrid screens and mass spectrometry (MS) analyses of affinity-purified Ecm29 complexes provide further support for the idea that Ecm29 is an adaptor in mammalian cells.…”

“…Proteins-Based on its intracellular distribution in HeLa cells we proposed that Ecm29 is an adaptor that recruits 26 S proteasomes to the endoplasmic reticulum and endosomal membranes as well as to the centrosome (30). If the adaptor hypothesis is correct, there should be proteins in these compartments that bind Ecm29.…”

“…Previously we showed that, although Ecm29 is stably associated with the 26 S proteasome after homogenization in 0.25 M sucrose, it dissociates from the 26 S enzyme in extracts containing 0.25% Triton X-100 (30). Detergent-mediated dissociation of Ecm29 has proved useful, because the Ecm29-interacting proteins listed in Fig.…”

The cellular response to heat stress in the gobyGillichthys mirabilis: a cDNA microarray and protein-level analysis

“…That for Ecm29, a protein associated with the proteasome and proposed to link secretory compartments involved in protein quality control to centers of proteolysis (Gorbea et al, 2004), was induced after 270·min, after which, levels of message remained high for the duration of the experiment. In addition, an unidentified gene coding for a protein containing a HECT domain was transiently induced in late stage recovery.…”

Muscle Wasting in Aged, Sarcopenic Rats Is Associated with Enhanced Activity of the Ubiquitin Proteasome Pathway

“…USP14 is a DUB, which also regulates the degradative capacity of the 26S (6,39). In yeast, Ecm29 was found to stabilize the 19S-20S association (40), and in mammalian cells, Ecm29 is largely found on vesicular structures of the endosomal pathway (41,42). PA200 (Blm10 in yeast) can form hybrid 26S proteasomes, 19S-20S-PA200, and has been implicated in DNA repair (43).…”