An N-terminal Ca²⁺-binding motif regulates the secretory pathway Ca²⁺/Mn²⁺-transport ATPase SPCA1

The Ca²⁺/Mn²⁺ transport ATPases 1a and 2 (SPCA1a/2) are closely related to the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) and are implicated in breast cancer and Hailey-Hailey skin disease. Here, we purified the human SPCA1a/2 isoforms from a yeast recombinant expression system and compared their biochemical properties after reconstitution. We observed that the purified SPCA1a displays a lower Ca²⁺ affinity and slightly lower Mn²⁺ affinity than SPCA2. Remarkably, the turnover rates of SPCA1a in the presence of Mn²⁺ and SPCA2 incubated with Ca²⁺ and Mn²⁺ were comparable, whereas the turnover rate of SPCA1a in Ca²⁺ was 2-fold higher. Moreover, we noted an unusual biphasic activation curve for the SPCA1a ATPase and autophosphorylation activity, not observed with SPCA2. We also found that the biphasic pattern and low apparent ion affinity of SPCA1a critically depends on ATP concentration. We further show that the specific properties of SPCA1a at least partially depend on an N-terminal EF-hand-like motif, which is present only in the SPCA1a isoform and absent in SPCA2. This motif binds Ca²⁺, and its mutation lowered the Ca²⁺ turnover rate relative to that of Mn²⁺, increased substrate affinity, and reduced the level of biphasic activation of SPCA1a. A biochemical analysis indicated that Ca²⁺ binding to the N-terminal EF-hand-like motif promotes the activity of SPCA1a by facilitating autophosphorylation. We propose that this regulation may be physiologically relevant in cells with a high Ca²⁺ load, such as mammary gland cells during lactation, or in cells with a low ATP content, such as keratinocytes.