RESUMO
Calpain 7 (also known as PalBH) is a mammalian homologue of the Aspergillus, atypical calpain PalB. Knowledge of the biochemical properties of calpain 7 is limited and its function is not yet known. In this study, we investigated the interactions of calpain 7 with all 11 ESCRT-III-related proteins, named charged multivesicular body proteins (CHMPs), and the subcellular localization of calpain 7. Pulldown assays using stable HEK293T transfectants of Strep-tagged calpain 7 revealed interactions of calpain 7 with a subset of FLAG-tagged CHMPs, among which CHMP1B was selected for further analyses. The N-terminal region containing a tandem repeat of MIT domains of calpain 7 was found to be necessary and sufficient for interaction with CHMP1B. Direct interaction was confirmed by a pulldown assay using recombinant proteins. Fluorescence microscopic analysis using HeLa cells revealed that overexpression of GFP-fused CHMPs or a dominant-negative construct of SKD1/Vps4B caused accumulation of epitope-tagged calpain 7 in a punctate pattern in the perinuclear area. Subcellular fractionation revealed that the most of endogenous calpain 7 is present in the cytosol but a small portion is present in particulate fractions. Punctate fluorescence signals of monomeric GFP-fused calpain 7 partly merged with those of endocytosed tetramethylrhodamine-labelled EGF. These results suggest that calpain 7 plays roles in the endosomal pathway by interacting with a subset of ESCRT-III-related proteins.
Assuntos
Calpaína/metabolismo , Membrana Celular/metabolismo , Endossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Calpaína/genética , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Plasmídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Frações Subcelulares , Proteínas de Transporte Vesicular/genéticaRESUMO
ALG-2 (apoptosis-linked gene 2) is a Ca2+-binding protein that belongs to the PEF (penta-EF-hand) protein family. Alix (ALG-2-interacting protein X)/AIP1 (ALG-2-interacting protein 1), one of its binding partners, interacts with TSG101 and CHMP4 (charged multivesicular body protein 4), which are components of ESCRT-I (endosomal sorting complex required for transport I) and ESCRT-III respectively. In the present study, we investigated the association between ALG-2 and ESCRT-I. By a GST (glutathione S-transferase) pull-down assay using HEK-293T (human embryonic kidney 293T) cell lysates, endogenous TSG101 and two other exogenously expressed ESCRT-I components [hVps28 (human vacuolar protein sorting 28) and hVps37A] were shown to associate with GST-ALG-2 in the presence of Ca2+. By the yeast two-hybrid assay, however, a positive interaction was observed with only TSG101 among the three ESCRT-I components, suggesting that ALG-2 associates with hVps28 and hVps37A indirectly through TSG101. Using various deletion mutants of TSG101, the central PRR (proline-rich region) was found to be sufficient for interaction with ALG-2 by the GST-pull-down assay. Direct binding of ALG-2 to the TSG101 PRR was demonstrated by an overlay assay using biotin-labelled ALG-2 as a probe. In immunofluorescence microscopic analysis of HeLa cells that overexpressed a GFP (green fluorescent protein)-fused ATPase-defective dominant-negative form of SKD1/Vps4B (GFP-SKD1(E235Q)), ALG-2 exhibited a punctate distribution at the perinuclear area and co-localized with GFP-SKD1(E235Q) to aberrant endosomes. This punctate distribution of ALG-2 was markedly diminished by treatment of HeLa cells with a membrane-permeant Ca2+ chelator. Moreover, a Ca2+-binding-defective mutant of ALG-2 did not co-localize with GFP-SKD1(E235Q). Our findings suggest that ALG-2 may function as a Ca2+-dependent accessory protein of the endosomal sorting machinery by interacting directly with TSG101 as well as with Alix.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Motivos EF Hand , Endossomos/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Proteínas Reguladoras de Apoptose/genética , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular , Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Genes Dominantes/genética , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas , Transporte Proteico , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Proteínas de Transporte VesicularRESUMO
Alix/AIP1 is a multifunctional adaptor protein involved in endocytosis, cell adhesion, and cell death. By yeast two-hybrid screening we identified a novel Alix/AIP1-interacting protein named Rab GTPase-activating protein-like protein (RabGAPLP). Interaction between Alix and RabGAPLP was confirmed by pull-down assays using fusion proteins of either glutathione-S-transferase (GST) or chitin-binding domain (CBD) and lysates of cultured mammalian cells expressing the respective proteins. Partial colocalization of FLAG-tagged RabGAPLP and green fluorescent protein (GFP)-fused Alix was observed at cell edges and filopodia-like structures by fluorescence confocal laser scanning microscopic analysis. The identity of RabGAPLP to merlin-associated protein (MAP), one of the interacting partners of neurofibromatosis type 2 (NF2) tumor suppressor gene product (merlin), implies cross-talk of membrane traffic and cell adhesion.